Magnetic nanosensor compositions and bioanalytical assays therefor

ABSTRACT

Disclosed are magnetic nanosensors or transducers that permit measurement of a physical parameter in an analyte via magnetic reasonance measurements, in particular of non-agglomerative assays. More particularly, in certain embodiments, the invention relates to designs of nanoparticle reagents and responsive polymer coated magnetic nanoparticles. Additionally provided are methods of use of nanoparticle reagents and responsive polymer coated magnetic nanoparticles for the detection of a stimulus or an analyte with NMR detectors.

FIELD OF THE INVENTION

This invention relates generally to NMR systems with magneticnanosensors for detection of analytes. More particularly, in certainembodiments, the invention relates to NMR-based analyte detectionsystems using responsive polymer-coated magnetic nanoparticles andnon-agglomerative bioanalytical assays.

BACKGROUND OF THE INVENTION

Biocompatible magnetic nanosensors have been designed to detectmolecular interactions in biological media. Upon target binding,magnetic nanosensors cause changes in spin-spin relaxation times ofneighboring water molecules (or any solvent molecule with freehydrogens) of a sample, which can be detected by classical magneticresonance (NMR/MRI) techniques. By using nanosensors in a sample, it ispossible to detect the presence of an analyte at very lowconcentration—for example, small molecules, specific DNA, RNA, proteins,carbohydrates, lipids, lipoproteins, organisms, and pathogens (e.g.bacteria, viruses, etc.)—with sensitivity in the low femtomole range(e.g., about 0.5 fmol to about 30 fmol per microliter; less than tenanalyte particles (e.g., virus/cell) per microliter).

In general, magnetic nanosensors used are superparamagneticnanoparticles functionalized with affinity moieties that bind orotherwise link to their intended molecular target to form clusters(aggregates) or nanoassemblies. It is thought that whensuperparamagnetic nanoparticles assemble into clusters and the effectivecross sectional area becomes larger, the nanoassembly becomes moreefficient at dephasing spins of surrounding water (or other solvent)protons, leading to an enhancement of measured relaxation rates (1/T2).Additionally, nanoassembly formation can be designed to be reversible(e.g., by temperature shift, chemical cleavage, pH shift, etc.) so that“forward” or “reverse” assays can be developed for detection of specificanalytes. Forward (clustering) and reverse (declustering) types ofassays can be used to detect a wide variety of biologically relevantmaterials. Furthermore, spin-lattice relaxation time (T1) is consideredindependent of nanoparticle assembly formation and can be used tomeasure concentration in both nano-assembled and dispersed states withinthe same solution.

Examples of magnetic nanosensors are described in Perez et al., “Use ofMagnetic Nanoparticles as Nanosensors to Probe for MolecularInteractions,” ChemBioChem, 2004, 5, 261-264, and in U.S. PatentApplication Publication No. US2003/0092029 (Josephson et al.), the textsof which are incorporated by reference herein, in their entirety.Examples of magnetic nanosensors include monocrystalline iron oxidenanoparticles from about 3 to about 5 nm in diameter surrounded with adextran coating approximately 10 nm thick such that the averageresulting particle size is from about 25 to about 100 nm. Anotherexample of magnetic nanosensors include polycrystalline iron oxidenanoparticles of about 100 nm to about 1 micron in diameter.

Nanosensors have demonstrated low femtomolar analyte detectionsensitivity through cluster formation (i.e. aggregation) and dispersion(i.e. disaggregation) assays. However, sensitivity is just onerequirement for a versatile bioanalytical detection system. A versatilebioanalytical detection system should also provide rapid results and beadaptable to functioning with a wide range of analyte concentrations fora variety of bioanalytical assays. Aggregation/disaggregation ofnanosensors may not be the optimal method for analyte detection in allassays.

For example, cluster formation can only occur when each nanoparticle isbound to multiple analytes and, in some cases, each analyte is bound tomultiple nanoparticles. Additionally, aggregation can be inhibited bygeometrical effects such as a variation in size among nanosensors andanalytes. Further, long incubation time may be required for clusterformation due to a two-step kinetic process for aggregation. Analyteneeds to first bind to one or more nanosensor(s), then nanosensorsagglomerate with each other to form clusters.

Cluster formation has also been shown to limit the dynamic range forcertain bioanalytical assays. Factors that may contribute to limitingdynamic range include the structural instability of clusteredaggregates. In addition, excess aggregation may lead to precipitation ofnanosensors out of solution. Further, imperfect magnetization couplingof nanoparticles with each other, over an extended period of incubationtime, may also contribute to a reduction in net magnetization per unitvolume of a cluster, making relaxation process less efficient andlowering its magnitude. Therefore, the need exists for designs ofversatile nanosensors and bioanalytical assays that exploit theindividual magnetic nanoparticle's enhanced capability of dephasingspins of water protons for analyte detection without aggregation.

SUMMARY OF THE INVENTION

Provided methods and compositions exploit the ability of magneticnanosensors to dephase nuclear spins, hereinafter generally exemplifiedas protons of water molecules, detectable by nuclear magnetic resonance(NMR) relaxation measurements (e.g., 1/T₂), for sensing and/or measuringan analyte of interest, without aggregation of nanosensors. Inprinciple, T₂ relaxivity of water protons dephased by nanosensors can beproportional to the diameter of nanosensors present in a solution andthe diffusion time of water protons in the proximity of individualnanosensors. Thus, provided compositions and bioanalytical assayscomprise magnetic nanosensors introduced into a sample that may containa target analyte in order to react with target analyte in a sample in arapid, and homogeneous reaction. Provided bioanalytical assays includecompositions and methods for optimizing the size or responsive size ofnanosensors, to enable rapid assay time-to-results, increasedsensitivity, and large (e.g., wide) relaxivity (e.g., T₂) dynamic range.

Provided are nanosensors that exploit the ability of magneticnanoparticles to dephase nuclear spins detectable by NMR, for detectionwithout aggregation of nanoparticles. In particular embodiments,provided nanosensors comprise a nanoparticle having a polymer matrixlayer which responds (e.g., expands or contracts) when exposed to ananalyte and/or a condition to be detected. A resulting change innanoparticle size affects dephasing of freely-diffusing water moleculesin the vicinity of the nanoparticles, which affects one or moreNMR-detectable properties.

In other particular embodiments, provided nanosensors comprise ananoparticle having a binding agent which is size optimized to provideoptimal T₂ measurement. A resulting change in nanoparticle size whenexposed to an analyte and/or a condition to be detected provides amaximum change in T₂.

Additionally provided are particular methods of using providedresponsive nanosensor compositions, wherein NMR-detectable propertiescan be obtained using responsive nanosensor compositions to determinethe existence and/or level of a condition and/or analyte of interest inone or more samples. In certain embodiments, comparing obtainedNMR-detected properties with a known control (e.g., one or morereference samples, a known control reference measure), may determine theexistence and/or level of a condition and/or analyte of interest in oneor more samples.

The objects and features of the invention can be better understood withreference to the drawings described below, and the claims. The drawingsare not necessarily to scale, emphasis instead generally being placedupon illustrating the principles of the invention. In the drawings, likenumerals are used to indicate like parts throughout various views.

While the present invention is particularly shown and described hereinwith reference to specific examples and specific embodiments, it shouldbe understood by those skilled in the art that various changes in formand detail may be made therein without departing from the spirit andscope of the invention.

BRIEF DESCRIPTION OF THE DRAWING OF THE INVENTION

FIG. 1 is a schematic depicting a principle of operation and elements ofa responsive polymer (e.g., matrix) coated nanosensor, according to anillustrative embodiment of the invention. FIG. 1 a is schematic of aregion of the matrix, depicting binding moieties of a responsive polymercoated nanosensor in one state (e.g., before stimulus). FIG. 1 b isschematic of a region of the matrix depicting binding moieties of aresponsive polymer coated nanosensor in another state (e.g., afterstimulus).

FIG. 2 is a schematic similar to FIG. 1, wherein the matrix takes theform of a membrane containing plural magnetic particles, according to anillustrative embodiment of the invention. FIG. 2 a is schematic of asmall region of the matrix depicting binding moieties of a responsivepolymer coated nanosensor in one state (e.g., before stimulus). FIG. 2 bis schematic of a small region of the matrix depicting binding moietiesof a responsive polymer coated nanosensor in another state (e.g., afterstimulus).

FIG. 3 is a schematic that depicts a principle of operation and deviceelements of a responsive polymer coated nanosensor for non-competitiveaffinity reactions, according to an illustrative embodiment of theinvention. FIG. 3 a is schematic of a region of a matrix depictingbinding moieties of a responsive polymer coated nanosensor fornon-competitive affinity reactions in one state (e.g., before stimulus).FIG. 3 b is schematic of a region of a matrix depicting binding moietiesof a responsive polymer coated nanosensor for non-competitive affinityreactions in another state (e.g., after stimulus).

FIG. 4 is a schematic that depicts a principle of operation and deviceelements of a responsive polymer coated nanosensor for competitiveaffinity reactions, according to an illustrative embodiment of theinvention. FIG. 4 a is schematic of a region of a matrix depictingbinding moieties of a responsive polymer coated nanosensor forcompetitive affinity reactions in one state (e.g., before stimulus).FIG. 4 b is schematic of a small region of a matrix depicting bindingmoieties of a responsive polymer coated nanosensor for non-competitiveaffinity reactions in another state (e.g., after stimulus).

FIG. 5 is a schematic that depicts a principle of operation and deviceelements of a responsive polymer coated nanosensor configured withmultiple binding moieties, according to an illustrative embodiment ofthe invention. FIG. 5 a is schematic of a small region of matrix,illustrating binding moieties of a responsive polymer matrix coatednanosensor configured with multiple binding moieties in one state (e.g.,before stimulus). FIG. 5 b is schematic of region of a matrix,illustrating binding moieties of a responsive polymer matrix coatednanosensor configured with multiple binding moieties in one state (e.g.,after stimulus).

FIG. 6 is a schematic that depicts a principle of operation and deviceelements for performing NMR measurements using responsive polymer matrixcoated nanosensors, and/or bioanalytical assays for analyte detectionaccording to illustrative embodiments of the invention.

FIG. 7 is a schematic that demonstrates the principle of operation anddevice elements for performing NMR measurements of responsive polymermatrix coated nanosensors for detection of a stimulus, according to anillustrative embodiment of the invention.

FIG. 8 is a schematic of an NMR system for detection of an echo responseof a layer containing one or more provided nanosensors attached orcoated onto one or more wall(s) of a device implant.

FIG. 9 is a schematic depicting a principle of operation and deviceelements of a smart device for sensing and control release of one ormore binding moieties.

FIG. 10 depicts theoretical and actual results of experimentsdetermining the effect of T2 measurements on nanosensor size. FIG. 10Adepicts the theoretical shift in T2 obtained by increasing the size of ananosensor by coating it with an antibody. As more antibodies aretitrated in to the reaction and more nanoparticles become saturated withantibody, measured T2 decreases in response to the increased size ofantibody-bound nanosensor. FIG. 10B depicts experimental results of T2measurements of identically prepared nanoparticles with increasinglevels of bound antibody or antibody conjugated to a large protein(alkaline phosphatase).

FIG. 11 depicts positive correlation between results obtained usingprovided methods and FIG. 11A depicts results demonstrative positivecorrelations between measured T2 and analyte concentrations for each ofprovided dispersive competitive assays and provided inhibitivecompetitive assays. FIG. 11B depicts a schematic of a two stepinhibitive assay as a preferred format for competitive assay method.

FIG. 12 depicts results of competitive nanosensor assays according tothe invention. FIG. 12A depicts results of dispersive competitiveassays; and FIG. 12B depicts results of inhibitive competitive assays.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

It is contemplated that devices, systems, methods, and processes of theclaimed invention encompass variations and adaptations developed usinginformation from the embodiments described herein. Adaptation and/ormodification of the devices, systems, methods, and processes describedherein may be performed by those of ordinary skill in the relevant art.

Throughout the description, where devices and systems are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are devices andsystems of the invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the invention that consist essentially of, or consist of, the recitedprocessing steps.

As used herein, an analyte (or target analyte) may include one or morespecies of one or more of the following: a protein, a peptide, apolypeptide, an amino acid, a nucleic acid, an oligonucleotide, atherapeutic agent, a metabolite of a therapeutic agent, RNA, DNA, anantibody, an organism, a virus, bacteria, a carbohydrate, apolysaccharide, and glucose. An analyte may also include, for example, alipid, a gas (e.g., oxygen, carbon dioxide), an electrolyte (e.g.,sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose,magnesium, phosphate, calcium, ammonia, lactate), a lipoprotein,cholesterol, a fatty acid, a glycoprotein, a proteoglycan, and/or alipopolysaccharide. Furthermore, as used herein, “detection of (an)analyte” may also mean measurement of physical properties of a solutioncontaining one or more analytes, for example, measurement of dipolemoment, ionization, solubility/saturation, viscosity, gellation,crystallization, and/or phase changes of the solution.

In certain embodiments, a parameter of the environment to be detected isthe concentration of one or more analyte(s) in the environment (e.g. ina liquid sample), where an analyte can be, for example, a protein,lipid, a gas (e.g., oxygen, carbon dioxide), an electrolyte (e.g.,sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose,magnesium, phosphate, calcium, ammonia, lactate), a lipoprotein,cholesterol, a fatty acid, a glycoprotein, a proteoglycan, alipopolysaccharide. a peptide, a polypeptide, an amino acid, a nucleicacid, an oligonucleotide, a therapeutic agent, a metabolite, ametabolite of a therapeutic agent, RNA, DNA, an antibody, an organism, avirus, a bacteria, a pathogen, a carbohydrate, a polysaccharide, and/orglucose. Alternatively, a parameter of the environment to be detectedmay be a static or dynamic pH or ionic strength of a sample solution.The parameter may also be a thermal, mechanical, electromechanical,electric, magnetic, acoustic, or optical stimulus. In other embodiments,the parameter is ionizing or non-ionizing radiation, an enzymaticreaction, a catalytic reaction, an acidic or basic stimulus, or a changein lipophilicity, hydrophobicity, or hydrophilicity.

As used herein, one or more binding moieties, binding pairs, or bindingpendants may broadly be a chemical binder, an electroactive mediator, anelectron-pair donor, and/or an electron-pair acceptor. A binding moietymay include one or more species of one or more of the following: anatom, an ion, a molecule, a compound, a catalyst, an enzyme, anelectroactive mediator, an electron-pair donor, an electron-pairacceptor, a lanthanide, an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent, a biological molecule, ametabolite of a therapeutic agent, a peptide, a polypeptide, a protein,a carbohydrate, a polysaccharide. Binding moieties may be a polymer, ormay be part of a polymer that is linked to magnetic particle.Alternatively, a binding moiety-target can be binding pairs, or bindingpendants, such as antibodies and cognate antigens. For example, abinding moiety-target may be avidin-biotin or a ligand binding proteinsuch as concanavalin A with affinity for a carbohydrate. Examples of thecombination of a binding moiety and a target thereto includecombinations of antigen and antibody, a certain saccharide and lectin,biotin and avidin, protein A and IgG, hormone and receptor thereof,enzyme and substrate, and nucleic acid and complementary nucleic acid orin each case, vice versa. In certain embodiments, binding moieties mayinclude one, two, or more types of oligonucleotides and/or one, two, ormore types of proteins, etc. Biological molecules include binding agentsor target molecules of biological origin or synthetically made moleculesthat mimic the performance of biological molecules. Examples include,but are not limited to, peptides with non-natural amino acids, peptidenucleic acids (PNA's), or natural or man-made organic molecules thatreact with specific sites on target biological molecules. In certainembodiments, the target analyte molecule is a nucleic acid, and eachbinding moiety includes one of two or more different oligonucleotides,wherein each oligonucleotide is complementary to a region on the targetnucleic acid that is different than the regions to which the otheroligonucleotides are complementary. In other embodiments, a targetmolecule can be a polypeptide, and each binding moiety includes one oftwo or more different antibodies, wherein each antibody specificallybinds to a binding site on the polypeptide that is different than thebinding sites to which the other antibodies bind. In some embodiments,binding moieties bind to each other including one or more antigen(s)with one or more antibody(ies) having affinity for the one or morespecific antigen(s). In some embodiment an antibody is a monoclonalantibody or antigen binding fragment thereof. A binding moiety caninclude a cleavage site that is selectively cleaved by a targetmolecule, and cleavage of the binding moiety results in separation ofthe cross-link. Alternatively, the binding moieties can be polypeptidesand the target molecule can be an enzyme. In other examples, eachbinding moiety can bind to another binding moiety to form a cleavagesite that is selectively cleaved by a target molecule, and cleavage ofthe binding moiety results in separation of the cross-link. Further, atarget can be a virus, virus components (i.e. capsids), a cell,components of cells (e.g., vescicles, apoptotic bodies, organelles, celldebris/dead cells), and other particles (e.g., circulating clots,cholesterol particles, plaques, forms of amyloid, and micelles). A cellcan be a prokaryotic cell such as bacteria or a eukaryotic cell such asmammalian cell including cells of a human organ. A target can be also bea surface antigen, a G-protein receptor, a polysaccharide, or any atom,ion, or molecule that antibody can be produced using known immunologicalmethods. Binding moieties preferably include functional groups forattachment to a substrate or surface. For example, the binding moietiescan include one or more species of one or more of the following: anamino group, a carboxyl group, a sulfhydryl group, an amine group, animine group, an epoxy group, a hydroxyl group, a thiol group, anacrylate group, and/or an isocyano group or a mixture thereof.

As used herein, “container” is understood to mean any localizer of aliquid sample, for example, a well, or an indentation, or a support, ora channel, or a reservoir, or a sunken volume, or a compartment, or arecessed area, or an enclosure with or without an opening, or a tube, ora trough. At least one surface of the container can be, but is notnecessarily, functionalized with one or more types of binding moieties.

As used herein, nanosensors, functionalized nanosensors or magneticnanosensors mean magnetic (e.g., paramagnetic, superparamagnetic)nanoparticles, optionally functionalized with one or more bindingmoieties. Each species of functionalized nanosensor used in acomposition or assay described herein can have unique characteristicsincluding the size of the nanosensors and the type of magneticmaterial(s) and coating used. Nanosensors can be functionalized withbinding moieties attached to their surfaces as described in furtherdetail herein. A magnetic core of the nanoparticles is preferablynanometer scale (for example, less than about 100 nm in diameter) and ispreferably paramagnetic or superparamagnetic. A core of thenanoparticles may be fabricated using known techniques from any type ofmagnetic, paramagnetic, or superparamagnetic nanometer-scale metal coreincluding an oxide and/or a hydroxide of Fe, Si, Sn, An, Ti, Bi, Zr,and/or Zn. Magnetic particles can be composed of single metal crystalsor of multiple metal crystals. Magnetic nanoparticles include, forexample, superparamagnetic particles, paramagnetic particles, and/ormagnetic particles, with sizes, for example, of less than about 1 μm inat least one dimension (e.g., diameter), less than about 500 nm in atleast one dimension (e.g., diameter), less than about 400 nm in at leastone dimension (e.g., diameter), less than about 300 nm in at least onedimension (e.g., diameter), less than about 200 nm in at least onedimension, 100 nm in at least one dimension (e.g., diameter), less thanabout 60 nm, less than about 50 nm, less than about 40 nm, less thanabout 30 nm, less than about 20 nm, less than about 10 nm, or less thanabout 5 nm in at least one dimension Magnetic nanoparticles used inparticular embodiments have a metal oxide core of about 1 nm to about 25nm, from about 3 to about 10 nm, or about 5 nm in diameter. Magneticparticles used in particular embodiments are superparamagnetic and havecrystalline core size from about 1 nm to about 100 nm.

Magnetic Resonance

A brief summary of the technical elements relating to the principles ofthe invention are provided herein. Nanosensors are introduced into asample, preferably a liquid such as water, which has an atomic nucleusthat has a non-zero spin, such as hydrogen. As is well known in the art,a magnetic component of such a nucleus becomes polarized or spatiallyoriented in a bias magnetic field, and may be induced into magneticresonance precession at the Larmor frequency. Magnetic components, ormagnetic moments, of nuclei are vector quantities and add together togive a resultant bulk magnetization vector that is the NMR signalmeasured by NMR spectrometers.

When in proximity of individual magnetic nanosensors, water protonsinteract with nanosensors and their magnetic field gradient throughtranslational and rotational diffusion, predominantly translational.Prolonged interaction of water protons with individual nanosensorstranslates to an enhanced dephasing of water protons, and thus changesin T₂ relaxivity. In general, in an NMR detector (e.g., a conventionalmagnetic relaxometer equipment or an apparatus specially designed for MRdetection), an external bias magnetic field is applied to a samplecomprising nanosensors. Before, during, and/or after a mixing and/orincubation period, one or more radio frequency pulses, preferably at orabout the Larmor frequency, are applied to stimulate water protons inthe proximity of nanosensors. T₂ relaxivity of nuclei can be detectedand processed and, in some embodiments, compared to a standard orcontrol sample. T₂ relaxivity measurements using MR detectors are usedto detect the nanosensors. Such measurements may allow for determinationof the presence and/or amount or concentration of a target analyte.

Following a perturbation such as that employed in recording NMR signals,a bulk magnetization vector recovers to its original steady state overtime. This process is referred to as nuclear magnetic relaxation. Thereare two fundamental time constants that can be measured for therelaxation process. Recovery of bulk magnetization along the directionof the bias magnetic field is described by spin-lattice relaxation timeor longitudinal relaxation time, designated as T₁. Typically, T₁ is onthe order of milliseconds to seconds. The single-exponential decay ofbulk magnetization in the plane perpendicular to the direction of thefirst magnetic field is described by the spin-spin relaxation time, ortransverse relaxation time, designated as T₂. For liquid signals, T₂ isgenerally in the range of 100 milliseconds or more.

A magnetic resonance measurement can be performed by applying one ormore RF (radio frequency) energy pulses to a sample and measuring bulkmagnetization that becomes reoriented by the pulse. RF pulses have afrequency equal to the Larmor frequency, and duration sufficient tocause a bulk magnetization vector to reorient into a plane perpendicularto the bias magnetic field, where the bulk magnetization vector (the NMRsignal) can be recorded over time. The most common method to measurespin-spin relaxation is that originally described by Carr and Purcelland later modified and known in the art as the Carr-Purcell modifiedMeiboom-Gill (CPMG) method.

A model can provide a framework for general working principles of theinvention, more specifically for quantifying and maximizing T₂relaxivity. A simplified nanoparticle is assumed to comprise a core(e.g., a spherical core) of superparamagnetic material, surrounded by ashell (e.g., a spherical shell) of non-magnetic material, all in water.The model can be applied or modified for use with nanoparticles of avariety of shapes, as well as for use with alternative solvents (e.g.,sample comprising hydrogen nuclei). Nanosensors in solution reduce a T₂time constant relative to plain water. The depolarization of a waterproton is due to a dipole magnetic field produced by a magnetized coreof nanosensors. Field distortion causes spins to process at differentfrequencies, leading to destructive interference. Although a CPMG methodnormally refocuses static field-nonuniformity effects, Brownian motionof water protons causes them to enter and exit field distortions in atime shorter than echo interval, thereby making spin dispersiontime-dependent and breaking the CPMG refocusing effect. The T₂relaxivity of water protons depolarized by nanosensors are proportionalto the specific volume fraction of nanosensors present in a solution,and the diffusion time of the water protons in the proximity ofindividual nanosensors. T₂ relaxivity observed for a solution in theproportionality is:

1/T₂α(V_(p))(R²/D)

where V_(p) is the specific volume fraction of the particles insolution, R the radius of the nanosensors particles, and D the diffusionconstant of water. The term R²/D is equal to diffusion time, τ_(d). Thisis the time duration for a water molecule to diffuse within the dipolemagnetic field produced by the magnetized core of the nanosensors, andis proportional to the extent of T₂ relaxation that occurs.

Certain aspects of the invention comprise bioanalytical assaysconfigured for rapid reaction kinetics and high dynamic range MRdetection. Provided bioanalytical assays can be used to assess thepresence and/or amount or concentration of a target analyte in a sample.

In certain embodiments, provided methods utilize superparamagneticnanosensors, a sample that may contain a target analyte, and a sample(e.g., an aqueous sample), which provides protons (e.g., hydrogenprotons) that emit magnetic resonance signals. In particular embodimentsa sample may be a fluid sample, including, e.g., water, saline, bufferedsaline, or a biological fluid. In certain embodiments a sample includesa biological fluid selected from one or more of blood, a cellhomogenate, a tissue homogenate, a cell extract, a tissue extract, acell suspension, a tissue suspension, milk, urine, saliva, semen, and/orspinal fluid.

Magnetic resonance signals can be influenced by diffusion, particularlydiffusion of water protons and diffusion of the nanosensors in a sample.Magnetic resonance measurements can also be influenced by spindiffusion, a phenomenon in which the spin or polarization of a nucleusis interchanged with that of a nearby nucleus of the same type. Spindiffusion can distribute spin-dependent effects, such as depolarization,throughout the sample. For example, if a small fraction of hydrogennuclei in water experience a depolarizing force, spin diffusion cancause all of the hydrogen in the sample to assume an averagedpolarization value.

In certain embodiments, nanosensors used in conjunction with providedmethods are superparamagnetic nanoparticles that produce highperturbations of the bias magnetic field in a region close to thenanoparticles. A paramagnetic or superparamagnetic core of ananoparticle becomes magnetized when an external bias magnetic field isapplied to it. A superparamagnetic core exhibits a high permeability,but little or no hysteresis. When placed in a magnetic field, a corebecomes strongly magnetized parallel to the direction of the appliedfield, then loses essentially all of its magnetization upon removal ofthe external magnetic field. A magnetized core produces a magnetic fieldwhich usually approximates a dipole field, or the magnetic fieldproduced by an ideal magnetic dipole located at the center of theparamagnetic core of the nanoparticle. The dipole field adds linearly tothe applied field (as vectors), resulting in a net magnetic field or netmagnetization. The Larmor frequency is determined by a net magneticfield experienced by polarized nuclei or water protons. In general,nanosensors, and derivatives thereof known in the art may be adaptedaccording to the description herein for production of compositions anduse in conjunction with provided methods.

In general, the functionalized nanosensors are contacted with a samplethat may contain a target analyte in a container. Either the originalcontainer containing the reagent(s) or a new container to which thereagents and sample have been transferred is placed within a RF coil ofa NMR detector. An RF frequency is tuned to the appropriate wavelengthas dictated by the strength of the magnetic field and gyromagnetic ratioof the detected nuclei. RF excitation pulses are product by aspectrometer and transferred to the RF coil which has been placed in theproximity of the container. These pulses, such as 90° or 180° pulses,generate signals, such as echoes, from the solvent (water) protons. Thedetected signal is influenced by the superparamagnetic nanoparticlesthat couple the magnitude or type of influence to the amount,concentration, or presence of the target analyte in the sample. Thepresence, amount and/or concentration of the target analyte in abiological fluid sample can then be determined from the detected RFsignal(s).

Provided aspects of the invention comprise bioanalytical nanosensorcoating assays configured for improved sensitivity, high dynamic rangeand improved diagnostic assay MR detection. Provided bioanalyticalassays can be used to assess the presence and/or amount and/orconcentration of analyte in a sample.

In prior work, methods of nanosensor based MR assays for detection ofanalyte comprise addition of reagents to a sample which results information of an ensemble of clusters of varying sizes, leading tomeasurable changes in relaxation rates (e.g., T2). Parameters of suchassays can be tuned to favor cluster formation of specific sizes. Suchapproaches have relied on reaching a dynamic equilibrium and kineticsduring nanoparticle self assembly; however, such methods can result inover titrated assay configurations, leading to nanoparticledestabilization (Kim et al, 2007; Taktak et al 2007; Lowery et al 2007;Kim et al 2008). Resulting destabilization can limit an overall dynamicrange of an assay, as well as lead to polydisperse mixtures ofnanoparticle clusters, which may decrease the overall change in T2relaxation rates in a sample, resulting in inaccurate detectionmeasurements. Additionally, in the classic MRSw design, resultingnanoparticle binding agent clusters can be of varying size. Control ofthe resulting solution (e.g., cluster formation, cluster stability),though possible, can be challenging. Under a given set of assayconditions (e.g., iron concentration, temperature, basic nanoparticlesize, diffusivity, inter-echo delay, etc), there is a decrease in T2signal as cluster size increases from <100 nm to ˜100 nm. Above ˜100 nm,T2 signal begins to rise. Therefore, when cluster formation isuncontrolled, it is difficult to ensure that a maximal ΔT2 signal isbeing achieved with a change in cluster size, as the change in size maybe moving the T2 in more than one direction, resulting in a decreasedabsolute ΔT2.

Provided methods comprise competitive assays, including competitivedispersive assays where analyte bound nanoparticles (e.g., nanoparticlesdecorated with analyte or analyte surrogate (e.g., analog)) arecomplexed in the presence of a binding agent (e.g., an antibody) priorto being challenged with sample; and inhibitive coating assays wheresample is pre-incubated with a binding agent (e.g., an antibody) priorto being challenged with analyte bound nanoparticles (e.g.,nanoparticles decorated with analyte or analyte surrogate (e.g.,analog)).

Provided methods comprise addition of a binding agent to a singlepopulation of analyte-bound nanoparticles. The method involves coating ananoparticle with a single layer of analyte, and a single layer ofbinding agent. Based on size measurements, the layer of binding agent isformed even if the binding agent is divalent (e.g., IgG antibodies).Addition of binding agent in this assay format leads to a uniformincrease in size among all nanoparticles. This size increase is onlydependent on concentration of binding agent and on the size of thebinding agent and analyte, which are both highly adjustable. Wehypothesize that this layer formation is thermodynamically andsterically favorable over cluster formation. Further, provided methodsresult in formation and assay of a single population of non-aggregatednanosensors, thereby allowing for optimized and accurate measurements ofrelaxation parameters.

Provided are methods wherein size of complexes formed upon addition ofbinding agent and/or target analyte is inherently limited by design ofthe assay reagents, and wherein thermodynamically and stericallyfavorable single, non-aggregated nanosensors coated with binding agentare formed. Provided methods reduce the polydispersity of nanosensorsolution after addition of analyte in favor of formation of bindingagent coated single nanosensor, thereby optimizing delta T2measurements. With this approach, complexes formed by addition ofanalyte are limited by the available binding sites and the size of thebinding agent, which is inherently easier to control and optimize ratherthan the thermodynamics and kinetics of the clustering process. Providedmethods allow the ability to directly control complex formation (anddetection measurements) through the architecture of assay reagents.

In certain embodiments, provided methods utilize superparamagneticnanosensors, one or more binding agent(s), and a sample (e.g., anaqueous sample) that may contain analyte and which provides protons(e.g., hydrogen protons) that emit magnetic resonance signals. Magneticresonance signals can be influenced by diffusion, particularly diffusionof water protons and diffusion of the nanosensors in a sample.

In certain embodiments, nanosensors are used in conjunction withprovided methods in a manner similar to those generally known in the artand generally described herein, with particular selection of reagents tooptimize for robust and controllable MR measurements through use ofprovided nanosensor coating assays. In general, nanosensors, andderivatives thereof known in the art may be adapted according to thedescription herein for production of compositions and use in conjunctionwith methods of measurement of relaxation. Additionally, oralternatively, provided transducers comprising nanosensors havingresponsive polymer coatings, as well as provided MRSw coating assaycompositions may be adapted (independently or in conjunction) for use inconjunction with the present methods.

In particular embodiments, nanosensors are functionalized with one ormore of a variety different types of binding moieties. Where more thanone functionalized nanosensors are used in an assay described herein,each one used will preferably be functionalized with a binding moietythat has affinity for a different target than the other(s).

In one embodiment, a bioanalytical assay for the detection of one ormore target analytes in a sample includes introducing one or morespecies of functionalized nanosensors into a sample, allowingfunctionalized nanosensors to interact with any target analyte(s) in thesample, measuring the change in T2 relaxivity and determining thepresence, amount and/or concentration of the target analyte(s). As aresult of interaction between binding moieties and target analyte,binding moieties bind to the target analyte, thereby producing (ordispersing) functionalized nanosensors comprising a monolayer coating ofbinding agent that partially or completely surrounds or coats thenanoparticle. As binding agent adds to or disperses from a monolayercoating of nanosensor(s), the effective size of the nanosensors change.These factors serve to produce a change in T₂ relaxivity that isproportional to an amount or concentration of the target analyte or thatindicates the presence of the target analyte.

In certain embodiments methods comprise “coating” analyte-decoratednanoparticles with binding agents of known and controlled size. The sizeof the binding agent (e.g., an antibody) can be optimized by theaddition of size increasing moieties of known size to a non-bindingregion of the binding agent. Optimization depends upon the sizes ofmagnetic nanoparticle being utilized, and any associated coating, aswell as the analyte coating and the binding agent. Because the bindingagent binds directly and exclusively to analyte coated particles, amaximum achievable diameter of the resulting particle is roughly 3 timesthe diameter of the individual nanoparticle, plus the lengths of thebinding agent and any size altering body attached to it. Final clustersize can therefore be controlled directly by varying the initialnanoparticle size, analyte size, as well as by varying the size of anysize modifying structure that may be attached to the binding agent. Thisgives the user direct control over the maximum finalnanoparticle-binding agent complex size to ensure an optimal ΔT2 signal.This level of control was not possible during the formation of branchingclustered structures formed using the traditional clustering method.

Certain embodiments comprise inhibitive coating assays where a sample ispre-incubated with a binding agent (e.g., an antibody) prior to beingchallenged with analyte bound nanoparticles (e.g., nanoparticlesdecorated with analyte or analyte surrogate (e.g., analog)). Preferablyreagents are selected for formation of nanosensors capable of generatinga maximal ΔT2 signal. This should significantly improve the dynamicrange and sensitivity of nanosensor assays requiring competitivedispersion to detect analyte.

Some embodiments comprise competitive dispersive coating assays whereinbound binding agent-analyte coated nanoparticle nanosensors arepre-formed. Preferably reagents are selected for formation ofnanosensors capable of generating a maximal ΔT2 signal. This shouldsignificantly improve the dynamic range and sensitivity of nanosensorassays requiring competitive dispersion to detect analyte.

Provided methods expand the number of potential assays that can bedeveloped. Thus, provided methods have the potential to greatly improvethe performance of a wide range of detection assays in terms ofsensitivity and dynamic range, facilitating faster development times andimproved diagnostic assay performance. In addition, competitivedispersive nanosensor assays can now be produced for target analytesthat lack multi-valent binding agents. Competitive dispersive MRSwassays may be useful, and in certain instances required, when developingdetection assays for monovalent targets. In certain embodiments themethod comprises use of single valence binding agents (e.g., monoclonalantibody).

In general, functionalized nanosensors are contacted with a sample thatmay contain a target analyte in a container. Either an originalcontainer containing the reagent(s) or a new container to which thereagents have been transferred is placed within a RF coil of a NMRdetector. An RF frequency is tuned to the appropriate wavelength asdictated by the strength of the magnetic field and gyromagnetic ratio ofthe detected nuclei. RF excitation pulses are product by a spectrometerand transferred to the RF coil which has been placed in the proximity ofthe container. These pulses, such as 90° or 180° pulses, generatesignals, such as echoes, from the solvent (water) protons. The detectedsignal is influenced by the superparamagnetic nanoparticles that couplethe magnitude or type of influence to the amount, concentration, orpresence of the target analyte in the sample. The presence, amountand/or concentration of the target analyte in a biological fluid samplecan then be determined from the detected RF signal(s).

Provided bioanalytical assays may be used to assess the presence, amountand/or concentration of a variety of different types of target analytes,including proteins, lipids, electrolytes and related clinical chemistryanalytes (e.g., sodium, potassium, chloride, bicarbonate, BUN,creatinine, glucose, magnesium, phosphate, calcium, ammonia, lactate),lipoproteins, cholesterol, fatty acids, glycoproteins, proteoglycans,lipopolysaccharides, peptides, polypeptides, amino acids, nucleic acids,oligonucleotides, therapeutic agents, metabolites, metabolites oftherapeutic agents, RNA, DNA, antibodies, organisms, viruses, viralcapsids, bacteria, pathogens, prions, carbohydrate such aspolysaccharides or monosaccharides, human cells, vesicles, apoptoticbodies, organelles, cell debris, cell clots, amyloid, micelles, andvarious other biological molecules. Biological molecules can be eithermolecules of natural biological origin or synthetically made moleculesthat can, in some way, mimic the performance of biological molecules.Examples of such synthetic biological molecules include peptides withnon-natural amino acids, peptide nucleic acids (PNA's), or natural orman-made organic molecules that react with specific sites on targetbiological molecules.

In certain embodiments, a target analyte can be a nucleic acid and thebinding moiety can be an oligonucleotide that is complementary to aregion on the target nucleic acid. In other embodiments, a targetanalyte can be a polypeptide and a binding moiety can be an antibodythat specifically binds to a binding site on the polypeptide.Alternatively, binding moieties can be polypeptides and a target analytecan be an enzyme. In yet another embodiments, provided bioanalyticalassays can be used for detection and/or differentiation of normal cells,abnormal cells (e.g., diseased cells, infected cell, tumor cells) andtheir subpopulation using binding moieties for specific surface markers.

In certain embodiments methods for detection of an analyte in a sampleare provided. In some embodiment, the method comprises providingnanosensors and providing a fluid sample. The nanosensors comprisemagnetic nanoparticles linked to an analyte or analog thereof. Thenanosensors further comprise one or more binding moieties optionallylinked to the analyte or analog thereof. The binding moieties areresponsive to the analyte or analog thereof bound to the nanoparticle,as well as to analyte present in the sample. The nanosensor includinganalyte or analog thereof linked to the nanoparticle and bindingmoiety(ies) bound to analog are size optimized to confer optimalrelaxation measurements. In some embodiments the method includes placingthe sample and the nanosensors in a container under conditions and for asufficient period of time to allow analyte in the sample to bind to andcompete off the binding moiety from the analyte or analog thereof on thenanosensor; placing the container in proximity to an NMR detector;measuring one or more relaxivity parameters of the sample in thecontainer; and determining one or more attributes relative to thesample.

In other embodiments methods for detection of an analyte in a sample areprovided comprising providing a fluid sample and one or more bindingmoieties, the binding moieties responsive to a target analyte or analogthereof and placing the sample and binding moieties under conditions andfor a sufficient period of time to allow analyte in the sample to bindto binding moiety. The methods further include providing nanosensorscomprising magnetic nanoparticles linked to an analyte or an analogthereof, which analyte is responsive to the binding moieties incubatedwith the sample, and placing the pre-incubated sample and bindingmoiety(ies) and the nanosensors in a container under conditions and fora sufficient period of time to allow analyte linked to nanosensors tobind to and compete off the binding moiety from the analyte in thesample then placing the container in proximity to an NMR detector. Onceplaced in the detector, one or more relaxivity parameters of the samplein the container are measured, and one or more attributes relative tothe sample are determined. The nanosensor including analyte or analogthereof linked to the nanoparticle and binding moiety(ies) bound toanalog are size optimized to confer optimal relaxation measurements.

In some embodiments the attribute of a sample which is assessed ordetermined is any one or more of the presence of the analyte, the amountof the analyte and/or the concentration of the analyte in a sample. Asdiscussed herein, in certain embodiment an analyte comprises at leastone member selected from the group consisting of a protein, a peptide, apolypeptide, an amino acid, a nucleic acid, an oligonucleotide, atherapeutic agent, a metabolite of a therapeutic agent, RNA, DNA, anantibody, an organism, a virus, a bacteria, a carbohydrate, and apolysaccharide.

In certain aspects, a family of transducers for placement in anenvironment, e.g., for contact with a solution, typically an aqueousanalyte solution are provided. When in an environment, providedtransducers develop or display a proton or other nuclear spin relaxivityproportional to a physical parameter of the environment, or a componentwithin the environment, which relaxivity is measureable from outside thesolution using a magnetic reasonance detection device. Thus, contact ofa transducer with an environment, such as a liquid sample containing ananalyte of interest, and optional incubation to promote establishment ofequilibrium between the environment and the transducer, permits indirectdetection of the value of a preselected physical (including chemical)property of the environment and/or determination of the presence and/orconcentration of a target molecule in the environment. The environmentmay be, for example, in vivo or ex vivo, where the environmentcontacting the transducer is a biological sample.

A transducer comprises one or more magnetic, e.g., paramagnetic orsuperparagmagnetic, particles having a polymer matrix layer containingone or more binding moieties. The binding moieties are responsive to thepresence or concentration of an analyte in the environment and/or areresponsive to some other property of the environment, such as, forexample, static pH, dynamic pH, and/or ionic strength of theenvironment. The polymer matrix layer may partially or completely coatthe magnetic core of each nanoparticle. The matrix may be, for example,a polymeric, hydrophilic, water-permeable hydrogel. In certainembodiments, the transducer operates by taking advantage of thedephasing of freely-diffusing water molecules in the vicinity of theresponsive matrix layer of the nanoparticles. In the presence of ananalyte or upon exposure to a condition to which the polymer matrixlayer is responsive, the specific volume of the polymer matrix layerchanges, leading to a detectable change in an NMR-measured property ofprotons in the environment of the nanoparticles(s). An NMR-measuredproperty may be, for example, T1 and/or T2 relaxivity. For example, achange in T2 may be related to (1) a change in the magnetic particlesize and (2) a change in the diffusion time of water (or otherproton-containing molecules) in the vicinity of the particle, relativeto particle size. Flux of water and/or other proton-containing moleculesout of or into the matrix may also affect T2, but this effect may not besignificant in light of (1) and (2). However, certain embodiments maytake advantage of the effect on T2 (or other NMR-measured property) ofthe change in flux of proton-containing molecules out of or into thematrix, for example, in embodiments where magnetic nanoparticle coresare embedded in responsive polymer matrix material.

The transducer may take many geometric and chemical forms. It may takethe form of a three dimensional mass of substrate material containingone or a plurality of polymer matrix-coated nanoparticles. The substratemay be in the form of a planar sheet or membrane. Preferably, thesubstrate does not significantly inhibit free diffusion of watermolecules (or other proton-containing environmental molecules) abouteach polymer-coated nanoparticle. In certain embodiments, thenanoparticles are immobilized on a substrate surface by means ofnonspecific absorption, specific chemical coupling, or specific bindingof nanoparticle to substrate material. In certain embodiments, part ofeach nanoparticle is immobilized on the substrate surface while anotherpart is free to interact with the sample environment (e.g., solution).The magnetic core of the nanoparticles are preferably nanometer scale(e.g., less than about 100 nm diameter, about 1 nm to about 100 nm) andare paramagnetic or superparamagnetic. The core of the nanoparticlesadvantageously may be fabricated using known techniques from any type ofmagnetic, paramagnetic, or superparamagnetic nanometer-scale metal coreincluding an oxide and/or a hydroxide of Fe, Si, Sn, An, Ti, Bi, Zr,and/or Zn. In particular embodiments magnetic particles comprise singlemetal crystals; in other embodiments magnetic particles comprisemultiple metal crystals. Magnetic nanoparticles preferably have a metaloxide core of about 1 to about 25 nm, from about 3 to about 10 nm, orabout 5 nm in diameter. The responsive coating matrix may have athickness in the range of 5 nm to 10,000 nm.

In a case where detected nuclei are water protons, a matrix preferablytakes the form of a stimuli or molecule sensitive hydrogel comprising apolymer “mesh” that is cross-linked by binding moieties that affects thevolume, permeability and the proton content of the matrix as a functionof a physical or chemical stimulus or a physical parameter of theanalyte under study. This is accomplished by design of the matrix as ahydrophilic polymer network comprising (as pendent groups or as part ofthe polymer backbone) binding moieties that influence water permeability(and/or permeability of other molecules in the environment) throughformation of one or more covalent or hydrogen bonds, van der Waalsinteractions, or physical entanglement with a component of the analyte.The presence of analyte induces a change in the crosslink density of thepolymer, which leads to a change in the volume fraction of the solutionoccupied by the polymer. The change in cross link density also leads toa change in the diameter of the nanoparticles, which leads to a changein their diffusion time. As discussed above, both diffusion time andspecific volume are proportional to the T₂ relaxivity observed for asolution, as shown in the proportionality:

1/T₂α(V_(p))(R²/D)

where V_(p) is the specific volume fraction of the particles insolution, R the radius of the particles, and D the diffusion constant ofwater. The term R²/D is equal to the diffusion time, τ_(d). This is thetime necessary for a water molecule to diffuse past a particle, and isproportional to the extent of T₂ relaxation that occurs.

As discussed above, a binding moiety may broadly be a chemical binder,an electroactive mediator, an electron-pair donor, and/or anelectron-pair acceptor. For example, the binding moiety may be an aceticacid moiety such as in poly(acrylic acid) for sensing pH, orphenylboronic acid for sensing the presence of diols, such as glucoseBinding moieties may be antibodies that serve as cross-linkers in thepresence of their cognate antigen, or antigens that serve ascross-linkers in the presence of their cognate antibodies, and whichmediate the water proton flux in and out of the matrix and change inspecific volume by competitive affinity reactions. This typically isaccomplished as the extent of cross-linking of matrix polymer ismediated as a function of the physical parameter under study so as tocontrol the permeability of water, including its amount and rate oftranslational diffusion in an out of the matrix and within the matrixvolume in proximity to the magnetic particle(s). For example, thebinding pairs may be a ligand binding protein such as concanavalin Abound to a low-affinity ligand such as a carbohydrate. Addition ofglucose to this system would displace the low affinity ligand and changethe crosslinking of the matrix. Another example is a matrix-immoblizedantibody, antibody fragment, or peptide that crosslinks the matrix bybinding to its matrix-immobilized antigen or target. The presence of ahigher affinity analyte would lead to disruption of the cross-linkedmatrix and a swelling of the matrix.

Accordingly, provided are responsive polymer coated magneticnanoparticle conjugates which behave as transducers and methods fortheir use. Each conjugate comprises a magnetic core covered at leastpartially with polymer matrix, e.g., a core embedded within a “matrix”or coated with a matrix or volume of water-permeable material whoselevel of association with nuclei and specific volume is responsive tothe value of the physical or chemical parameter under study. Theresponsive matrix may comprise a matrix of material which includes oneor more monomers and/or polymers. The one or more monomers and/orpolymers contain functional groups that enable the binding moiety to beattached to or otherwise in stable association with the nanoparticle toform the conjugate. The polymer can be a natural polymer, a syntheticpolymer, a combination of natural and synthetic polymers, shape memorypolymers, block co-polymers (PEO, PPO), or derivatives of each type. Forexample, the matrix polymer may be poly (N-isopropylacrylamide). Thematrix polymer may also be (or include), for example,Poly(N-isopropylacrylamide) (PNIAAm), Poly(N,N-diethyacrylamide)(PDEAAm), P(NIAAm-co-BMA), PEO-PPO-PEO (e.g., Pluronic®),N,N-diethylaminoethyl methacrylate (DEA), 2-hydroxypropyl methacrylate(HPMA), Poly-(methacrylic acid-g-ethylen glycol), Poly(-glucosyloxyethylmethacrylate), Poly(N-vinyl-2pyrrolidone-co-3-(acrylamido)phenylboronicacid), and/or N-(S)-sec-butylacrylamide. Functional groups may compriseone or more appropriate chemical functional group(s), e.g. carboxy,amino, or sulfhydryl groups. A specific moiety or moieties may beattached to the nanoparticle via conjugation to these groups, or byphysical adsorption and/or through hydrogen bonds or van der Waalsinteractions. The responsive polymer matrix, through physical and/orchemical stimuli, mediates the specific volume of the polymer layer,leading to a detectable change in NMR-measurable properties such as T₂relaxivity. The NMR-measurable property(ies) may be related to theparameter(s) under study via one or more calibration curves usingstandards of known parameter value. The NMR-measurable property(ies) mayinclude one or more discrete, continuous, differential, or integralmeasurements of the NMR relaxation rates (1/T₁, 1/T₂) and may bemeasured with an NMR detector of the solute or solvent withsusceptibility-induced dephasing by the magnetic nanoparticles. Inparticular embodiments a transducer conjugate can be used to measure anyphysical or chemical stimuli applied to the responsive polymer matrix.For example, the conjugate may be used for an assay for detecting aspecific target molecule, such as a biological molecule in a samplesolution or in vivo, by attaching one or more affinity moiety(ies)within the responsive polymer matrix with specificity for the targetmolecule. The conjugate may be used as a temperature sensor wherebythermal changes of the responsive polymer matrix result in changes ofsolvent flux or rate of change of flux as measured by changes in NMRrelaxation rates (1/T₁, 1/T₂). Thus, the new conjugate can be consideredto be a responsive polymer matrix coated superparamagnetic nanosensor ornanotransducer outputting a relaxivity detectable by the antenna of arelaxometer that is related to the value of the parameter underinvestigation. In certain embodiments a responsive polymer matrix coatedsuperparamagnetic nanosensor functions as a single entity, as a group ofnanosensors; in other embodiments a responsive polymer matrix coatedsuperparamagnetic nanosensor functions in an array of nanosensors, or inan encapsulation of nanosensors. Provided conjugate(s) may beincorporated with all embodiments and use for the detection of any of awide variety of analytes as disclosed in copending U.S. patentapplication Ser. No. 11/513,503, filed Aug. 31, 2006, titled NMR DEVICEFOR DETECTION OF ANALYTES, the disclosure of which is incorporatedherein by reference.

In one embodiment, each conjugate comprises a magnetic nanoparticlecoated with a responsive matrix. The responsive matrix may be, forexample, a polymeric, hydrophilic, water-permeable hydrogel. Theresponsive matrix contains one or more monomers or one or more polymers.The one or more monomer or one or more polymers contains one or morebinding moieties, as described above, that determine the physicalproperties of the matrix including porosity and permeability through oneor more covalent bonds, hydrogen bonds, van der Waals interactions, orphysical entanglement of one or more binding moiety(ies). The one ormore binding moiety(ies) mediates a swelling or shrinking of the polymermatrix, thereby leading to a change in T₂ by changing the specificvolume of the particles or by changing the radius of the particles andthereby the diffusion time of the nuclei, and/or mediates a change influx transport of solutes and solvent in and out of the responsivematrix; and, within the proximity of the magnetic nanoparticle and itsmagnetic field, magnetic field gradient when exposed to a magneticfield. The changes in matrix volume and/or changes in flux of solute orsolvent, in and out of the responsive matrix, subsequently in and out ofthe magnetic field, magnetic field gradient of the nanoparticle, isproportional to the NMR relaxation rates (1/T₁, 1/T₂) of the solute orsolvent with susceptibility-induced dephasing by a magneticnanoparticle. One or more discrete, continuous, differential, orintegral NMR measurements are used to quantify one or more physicalparameters, listed above, applied to the responsive matrix coatedmagnetic nanoparticle. Thus the responsive matrix coated magneticnanoparticle is a nanosensor or a transducer for the applied physical orchemical stimuli (e.g., optical sensor, pressure sensor, etc.). Thenanosensor may function as a single transducer for a specific stimulusor function simultaneously as a multiplex transducer with one or morenanosensors having sensitivities to different stimuli.

In another embodiment, magnetic nanoparticles each with one or moreresponsive polymer matrix layers are immobilized into or onto asubstrate either randomly or in an ordered array. The responsive polymermembrane is a hydrogel that contains cross-linked polymers, cross-linkedpolymers attached to magnetic particles, and magnetic particles havingpolymer matrices with cross-linking binding moieties. The constituentsof a hydrogel membrane including the cross-linking binding moietiesdetermines its physical properties including porosity and permeabilitythrough one or more covalent bonds, hydrogen bonds, van der Waalsinteractions, or physical entanglement of one or more bindingmoiety(ies). The one or more binding moiety(ies), previously listed,mediates the flux transport of solutes and solvent of a solution in andout of a hydrogel membrane and within the proximity of one or moremagnetic nanoparticle(s) and its (their) magnetic field(s), magneticfield gradient(s) when exposed to a magnetic field, effectively changingthe particle's volume fraction. The shrinking or swelling of the polymermatrix, which leads to a change in specific volume or diffusion time ofwater and/or the changes in flux of solute or solvent, in and out of thepolymer matrix, subsequently in and out of the magnetic field, magneticfield gradient of one or more nanoparticle(s) is proportional to the NMRrelaxation rates (1/T₁, 1/T₂) of the solute or solvent withsusceptibility-induced dephasing by one or more magneticnanoparticle(s). One or more discrete, continuous, differential, orintegral NMR measurement(s) is (are) used to quantify the physical orchemical stimuli applied to a hydrogel membrane thus providing atransducer for the applied physical or chemical stimuli (e.g., opticalsensor, pressure sensor, flow sensor etc.). A hydrogel membrane mayfunction as a transducer for a specific stimulus or function as amultiplex transducer by incorporating one or more magnetic particlesfunctionalized with sensitivities to different stimuli.

In another embodiment, each conjugate comprises a magnetic nanoparticlecoated with a responsive polymer matrix. The responsive polymer matrixis a hydrogel that contains cross-linked polymers. A hydrogel containsone or more cross-linking binding moieties that are responsive to the pHor ionic strength or changes in pH or ionic strength of the solution, orthe environment within a hydrogel, affecting one or more covalent bonds,hydrogen bonds, van der Waals interactions, or physical entanglement ofa hydrogel. The one or more binding moiety(ies) responsive to the pH orionic strength of the solution mediates the flux transport of solutesand solvent in and out of the hydrogel and within the proximity of themagnetic nanoparticle and its magnetic field, magnetic field gradientwhen exposed to a magnetic field. The shrinking or swelling of thepolymer matrix, which corresponds to a change in specific volume anddiffusion time, and/or the changes in flux of solute or solvent, in andout of the polymer coating, subsequently in and out of the magneticfield, magnetic field gradient of the nanoparticle is proportional tothe NMR relaxation rates (1/T₁, 1/T₂) of the solute or solvent withsusceptibility-induced dephasing by a magnetic nanoparticle. One or morediscrete, continuous, differential, or integral NMR measurements areused to quantify the pH or ionic strength of a solution containing oneor more of the hydrogel coated magnetic nanoparticles.

In another embodiment, each conjugate comprises a magnetic nanoparticlecoated with a responsive polymer matrix. The responsive polymer matrixis a hydrogel that contains cross-linked polymers. The hydrogel containsone or more binding moieties that generate a change in the pH or ionicstrength of a solution, or the environment within the hydrogel,affecting one or more covalent bonds, hydrogen bonds, van der Waalsinteractions, or physical entanglement of the hydrogel, as a resultantof reaction with one or more specific analyte(s) present in a samplesolution. The change in pH or ionic strength of the environment withinthe hydrogel mediates the flux transport of solutes and solvent in andout of the hydrogel and within the proximity of the magneticnanoparticle and its magnetic field, magnetic field gradient whenexposed to a magnetic field. The shrinking or swelling of the matrix,which correspond to a change in specific volume and/or diffusion time ofthe particles, and/or changes in flux of solute or solvent, in and outof the polymer coating, subsequently in and out of the magnetic field,magnetic field gradient of the nanoparticle is proportional to the NMRrelaxation rates (1/T₁, 1/T₂) of the solute or solvent withsusceptibility-induced dephasing by a magnetic nanoparticle. One or morediscrete, continuous, differential, or integral NMR measurements areused to quantify the amount or concentration of one or more analyte(s)present in the sample solution containing the hydrogel coated magneticnanoparticles.

In another embodiment, each conjugate comprises a magnetic nanoparticlecoated with a responsive polymer matrix. The responsive polymer matrixis a hydrogel that contains cross-linked polymers. The hydrogel containsone or more cross-linking binding moieties, or binding pairs, or bindingpendants that determines its physical properties including porosity andpermeability through one or more covalent bonds, hydrogen bonds, van derWaals interactions, or physical entanglement of one or more bindingmoieties, binding pairs, or binding pendants. The one or more bindingmoieties, binding pairs, or binding pendants mediates the shrinking orswelling of the polymer matrix and/or the flux transport of solutes andsolvent in and out of the hydrogel and within the proximity of themagnetic nanoparticle and its magnetic field, magnetic field gradientwhen exposed to a magnetic field, through one or more competitiveaffinity reactions. The change in specific volume arising from shrinkingor swelling and/or the flux or changes in flux of solute or solvent, inand out of the polymer coating, subsequently in and out of the magneticfield, magnetic field gradient of the nanoparticle is proportional tothe NMR relaxation rates (1/T₁, 1/T₂) of the solute or solvent withsusceptibility-induced dephasing by a magnetic nanoparticle. One or morediscrete, continuous, differential, or integral NMR measurements are useto quantify one or more analyte(s) present in the sample solutioncontaining the hydrogel coated magnetic nanoparticles, the one or moreanalyte(s) that binds one or more binding moiety(ies), binding pairs, orbinding pendants contained in the hydrogel.

In another embodiment, each conjugate comprises a magnetic nanoparticlecoated with a responsive polymer matrix. The responsive polymer matrixis a hydrogel that contains cross-linked polymers. The hydrogel containsone or more cross-linking binding moieties, or binding pairs, or bindingpendants that determines its physical properties including porosity andpermeability through one or more covalent bonds, hydrogen bonds, van derWaals interactions, or physical entanglement of one or more bindingmoieties, binding pairs, or binding pendants. The one or more bindingmoieties, binding pairs, or binding pendants, listed above withalternative embodiments, mediates the flux transport of solutes andsolvent in and out of the hydrogel and within the proximity of themagnetic nanoparticle and its magnetic field, magnetic field gradientwhen exposed to a magnetic field, through one or more non-competitiveaffinity reactions. The shrinking or swelling of the particle matrixand/or the flux or changes in flux of solute or solvent, in and out ofthe polymer coating, subsequently in and out of the magnetic field,magnetic field gradient of the nanoparticle, is proportional to the NMRrelaxation rates (1/T₁, 1/T₂) of the solute or solvent withsusceptibility-induced dephasing by a magnetic nanoparticle. One or morediscrete, continuous, differential, or integral NMR measurements areused to quantify one or more analyte(s) present in the sample solutioncontaining the hydrogel coated magnetic nanoparticles, the one or moreanalyte(s) that binds one or more binding moiety(ies), binding pairs, orbinding pendants contained in the hydrogel.

In another embodiment, each conjugate comprises a magnetic nanoparticlecoated with a responsive polymer matrix. The responsive polymer matrixis a hydrogel that contains cross-linked polymers. The hydrogel containsone or more cross-linking reactive moiety(ies) that determines itsphysical properties including porosity and permeability through one ormore covalent bonds, hydrogen bonds, van der Waals interactions, orphysical entanglement of the one or more reactive moiety(ies). The oneor more reactive moiety(ies) through a reaction with a reagent presentin a sample solution mediates the flux transport of solutes and solventin and out of the hydrogel and concomitant shrinking or swelling of thematrix that leads to a volume change within the proximity of themagnetic nanoparticle and its magnetic field, magnetic field gradientwhen exposed to a magnetic field, through one or more affinityreactions. The flux or changes in flux of solute or solvent, in and outof the polymer coating, subsequently in and out of the magnetic field,magnetic field gradient of the nanoparticle, and/or the change inspecific volume of nanoparticles is proportional to the NMR relaxationrates (1/T₁, 1/T₂) of the solute or solvent with susceptibility-induceddephasing by a magnetic nanoparticle. One or more discrete, continuous,differential, or integral NMR measurements are used to quantify one ormore analyte(s) present in the sample solution containing the hydrogelcoated magnetic nanoparticles, the one or more analyte(s) that reactswith one or more reactive binding moiety(ies) contained in the hydrogel.

The one or more reactive binding moiety(ies) of the responsive polymertransducers and methods of use described above may include one or morespecies of one or more binding moieties. For example, in one embodiment,the binding moieties may include one, two, or more types ofoligonucleotides and/or one, two, or more types of proteins. Bindingmoieties may be a polymer, or may be part of a polymer that is linked tomagnetic particle. The one or more reactive moiety(ies) may participatein a reaction resulting in a single product or participate in a reactiongenerating a cascade of reaction products. For example, a reactivemoiety may be glucose oxidase, and in the presents of glucose, areaction produces hydrogen peroxide and gluconic acid. The presence ofhydrogen peroxide or gluconic acid changes the pH of the environmentwithin the hydrogel or affect the extent of cross-linking of thehydrogel causing a reversible change in its permeability to solvent. Ingeneral one or more reactive moiety(ies) can react as to amplify theeffect of changes in the physical or chemical properties of the hydrogeland subsequently changes in its permeability to solute and solventwithin proximity of the magnetic nanoparticle and its magnetic field,magnetic field gradient when exposed to a magnetic field.

In still another aspect, the invention provides methods of determiningthe value of a physical parameter in a particular environment such as ananalyte solution (e.g., a liquid sample). The method comprises the stepsof exposing the transducer (nanoparticles with polymer matrix layer) ofany of the types disclosed herein to the environment, and allowing achange in crosslink density of the matrix to occur in accordance withthe environment (e.g., allowing equilibration of the matrix layer withthe surrounding liquid sample, where the sample contains an analyte towhich the matrix is responsive). Next, an external magnetic field isapplied to the particle, and radio frequency pulse, preferably at orabout the Larmor frequency, is applied to stimulate nuclei within thematrix adjacent the particle. Next, T₁ and/or T₂ relaxivity of thenuclei is detected, using ,e.g., conventional magnetic relaxometerequipment or apparatus specially designed for the purpose, and thedetected relaxivity signal is compared to a standard to determine thevalue of the parameter under investigation (e.g. via calibrationcurve(s) relating T1 and/or T2 relaxivity to the parameter underinvestigation, such as analyte presence or concentration in a liquidsample). The standard can take many specific forms, but may begenerically described as a data set relating the relaxivity parameter tospecific values for the parameter under study. It typically will bedeveloped using physical or chemical stimuli or analytes having knownspecific parameter values and corresponding relaxivity readings. In someembodiments, e.g., where the researcher or technician or clinician seeksto determine the presence of some particular molecule, the standard maybe merely a threshold value selected in the context of the performancegoals intended for the system in use. In this regard, in a preferredembodiment the methods may be used to assess the presence and/orconcentration of a molecule in an analyte solution, e.g., a biomoleculepresent in a biological fluid.

Provided responsive polymer matrix coated superparamagnetic nanosensorsmay be used for assessing the presence and/or concentration of one ormore analyte in a biological fluid. The nanosensors are exposed to abiological fluid sample contained within a container (e.g., well) andthe container (e.g., well) placed within a RF coil of an NMR detector.An RF excitation is applied to the container (e.g., well) at theappropriate wavelength, such wavelength being a calculable function ofmagnetic field strength and detected nuclei. The RF excitation producesone or more detectable RF signals, such as echoes, generated by thesolvent (water) protons within magnetic field, magnetic field gradientof superparamagnetic nanoparticles, within the container (e.g., well),which is a function of the concentration or presence of the analyte inthe fluid sample. The presence and/or concentration of the analyte ofbiological fluid sample can then be determined from the detected RFsignal(s).

Provided responsive polymer matrix coated superparamagnetic nanosensorsmay be used as a transducer for sensing a physical or chemical stimulus.The transducer may comprise single responsive polymer matrix coatedsuperparamagnetic nanosensor, as a group of nanosensors, an array ofnanosensors, or in an encapsulation of nanosensors. The transduceroperates in combination with an RF coil of an NMR detector. An RFexcitation is applied to the transducer at the appropriate wavelength,such wavelength being a calculable function of magnetic field strengthand detected nuclei. The RF excitation produces one or more detectableRF signals, such as echoes, generated by responsive polymer coatedsuperparamagnetic nanosensors within the transducer. The physical orchemical stimulus can then be determined from the detected RF signal(s).

Provided responsive polymer matrix coated superparamagnetic nanosensorsmay be used for coating, e.g., in the form of a hydrogel membrane, oneor more surface(s) of biosensors, medical devices, tools, instruments,and in vivo implants thus enabling smart or responsive devices. Inparticular embodiments, provided smart devices are used for the sensingof or within a biological cell, an organ, or biological fluids, oranalytes within a biological cell, an organ, or biological fluids. Incertain embodiments smart devices are used to sense the environmentsurrounding a device, a tool, an instrument, or an implant, or sense thephysical properties and conditions of a device, a tool, an instrument,or an implant itself. The transducer coating operates in combinationwith an RF coil of an NMR detector. An RF excitation is applied to thetransducer coating at the appropriate wavelength, such wavelength beinga calculable function of magnetic field strength and detected nuclei.The RF excitation produces one or more detectable RF signals, such asechoes, generated by responsive polymer coated superparamagneticnanosensors of the transducer coating or membrane. Further, in certainembodiments, one or more of the following: an atom, an ion, a molecule,a compound, a catalyst, an enzyme, an electroactive mediator, anelectron-pair donor, an electron-pair acceptor, a lanthanide, an aminoacid, a nucleic acid, an oligonucleotide, a therapeutic agent, abiological molecule, a metabolite of a therapeutic agent, a peptide, apolypeptide, a protein, a carbohydrate, a polysaccharide are loaded intoone or more coated nanosensor(s) within the responsive hydrogel or theresponsive hydrogel membrane itself and released in an opened loop orclosed-loop controlled manner by a selective physical, chemical, oranalyte stimulus applied to the responsive hydrogel membrane.

In FIG. 1, a principle on which the invention is based is explained onthe basis of one embodiment, and serves not to limit the invention to aparticular embodiment but for the purpose of explaining the principle.FIG. 1 shows device 100 which is a magnetic particle 101 coated with aresponsive polymer matrix 102. Polymer matrix 102 is permeable tosolvent molecule(s) 103. An example of solvent molecule 103 is watercontaining water protons. The responsive polymer matrix 102 containscross-linked polymers.

FIG. 1 a is a magnified cartoon representation of a small volume ofresponsive polymer matrix 102 in one state. Responsive polymer matrix102 can have one or more binding moiety(ies). FIG. 1 a depictsresponsive polymer matrix 102 with binding moiety 105 cross-linked withbinding moiety 106. Binding moiety 105 and 106 may be one or morepolymer chains of the responsive polymer matrix 102 or atoms, ions,molecules, or compounds attached to a backbone of the responsive polymermatrix 102. Binding moiety 105 and binding moiety 106 are cross-linkedthrough one or more covalent bonds, hydrogen bonds, van der Waalsinteractions, or physical entanglement of polymer chains represented byelement 107. The extent of cross-linking through element 107 definesphysical characteristics of a responsive polymer matrix 102, includingporosity, swelling, de-swelling, volume fraction, and permeability.Responsive polymer matrix 102 can be a hydrogel. The extent ofcross-linking of responsive polymer matrix 102 changes the specificvolume fraction of device 100 leading to a change in diffusion time ofsolvent molecules 103 in the proximity of magnetic particle 101.Magnetic particle 101 is capable of dephasing spins of solventmolecule(s) 103, and dephasing is measurable using NMR detection of NMRrelaxation rates (e.g., 1/T1, 1/T2). The specific volume change ofdevice 100 and change in diffusion time of solvent molecule(s) 103 isproportional to the NMR relaxation rates (e.g., 1/T1, 1/T2) of a solventmolecule 103, with susceptibility-induced dephasing by magneticnanoparticle 101. One or more discrete, continuous, differential, orintegral NMR measurements can be used to quantify the magnitude oramount of stimulus 104.

In FIG. 1, a responsive polymer matrix 102 is configured with one ormore binding moieties 105,106 as shown in FIG. 1 a with sensitivity andspecificity to a physical or chemical stimulus 104. Binding moieties105,106 may include one or more species of one or more of the following:an atom, an ion, a molecule, a compound, an electroactive mediator, anelectron-pair donor, an electron-pair acceptor. Binding moieties may bea polymer, or may be part of a polymer that is linked to magneticparticle. Binding moieties include functional groups, for example,binding moieties may include one or more species of one or more of thefollowing: an amino group, a carboxyl group, a sulfhydryl group, anamine group, an imine group, an epoxy group, a hydroxyl group, a thiolgroup, an acrylate group, and/or an isocyano group. A physical orchemical stimulus 104 may be one or more physical or chemical stimuliinclude(s) thermal, mechanical, electromagnetic energy,electro-mechanical, electric field, electromotive force, magnetic field,magnetic force, magnetic gradient force, electromagnetic force,photoacoustic energy, photoacoustic forces, electromagnetic radiation,non-ionizing radiation, ionizing radiation, enzymatic reactions,catalytic reactions, acidic stimulus, and basic stimulus, pH change,changes in ionic strength, lipophilicity, hydrophobicity, and/orhydrophilicity.

When stimulus 104 is applied to device 100, the cross-linking ofresponsive polymer matrix 102 through one or more covalent bonds,hydrogen bonds, van der Waals interactions, or physical entanglement ofthe polymer chains represented by element 107 changes in a manner shownin FIG. 1 b. FIG. 1 b is a magnified depiction of responsive polymer 102following transition into a responsive polymer matrix 109 upon receivingstimulus 104. Stimulus 104 causes element 107 to transition to element108, whereby the interaction between binding moiety 105 and bindingmoiety 106 reduces the extent of cross-linking thus transforming polymermatrix 102 into responsive polymer 109. Responsive polymer 109 will bemore permeable to solvent molecule(s) 103, thus enhancing the abilityfor solvent molecule(s) 103 to diffuse in and out of the responsivepolymer 109 and similarly in and out of the magnetic field, the magneticfield gradient of magnetic particle 101. In some embodiments aresponsive polymer of device 100 can be reversible upon cessation of thestimulus 104. In the absence of stimulus 104, binding moiety 105 canre-establish cross-linking with binding moiety 106, causing exclusion ofsolvent molecule(s) 103 from within the magnetic field, the magneticfield gradient of the magnetic particle 101, and subsequently from theresponsive polymer matrix. Magnetic particle 101 is capable of dephasingspins of lesser proportion of solvent molecule(s) 103 within responsivepolymer matrix 102 and the dephasing is measurable using NMR detectionof NMR relaxation rates (1/T1, 1/T2). The magnitude or amount ofstimulus 104 is proportional to NMR relaxation rates (1/T1, 1/T2) ordelta T2 of NMR relaxation rates (e.g., of solvent molecules(s) 103).Thus, device 100 is a sensor or transducer for stimulus 104. Themagnitude or amount of stimulus 104 can be quantified by proportionusing one or more discrete, continuous, differential, or integralmeasurement(s) of NMR relaxation rates (e.g., 1/T1, 1/T2) of solventmolecules(s) 103 with an NMR detector. Device 100 can be calibrated formeasurement of stimulus 104 by collecting a data set of NMR relaxationrates proportional to a predetermined range or standard of magnitude oramount of stimulus 104 and determining one or more calibration factors.One or more calibration factors will then be used to relate NMRrelaxation rates to specific values for a stimulus 104.

FIG. 2 shows device 200 which is a responsive polymer membrane 202containing magnetic particle(s) 201. Responsive polymer membrane 202 maybe fabricated by combining one or more device 101 through self-assemblyor fabrication methods that result in a sol-gel. A responsive polymermembrane 202 is permeable to solvent molecule(s) 203. An example ofsolvent molecule 203 is water containing water protons. Solvents highlysusceptible to dephasing by magnetic particles may be use in lieu ofwater 203. A responsive polymer membrane 202 contains cross-linkedpolymers.

FIG. 2 a is a magnified representation of responsive polymer membrane202 in one state. Responsive polymer membrane 202 can have one or morebinding moiety(ies). FIG. 2 a shows a responsive polymer membrane 202with binding moiety 205 cross-linked with binding moiety 206. Bindingmoiety 205 and 206 may be one or more polymer chains of a responsivepolymer membrane 102 or atoms, ions, molecules, or compounds attached toa backbone polymer of a responsive polymer membrane 102. Binding moiety205 and binding moiety 606 are cross-linked through one or more covalentbonds, hydrogen bonds, van der Waals interactions, or physicalentanglement of a polymer chains represented by element 207. The extentof cross-linking through element 207 defines physical characteristics ofa responsive polymer membrane 202 including porosity, swelling,de-swelling, volume fraction, and permeability. The extent ofcross-linking of responsive polymer membrane 202 controls permeabilityof solvent molecule(s) 203 including its (their) amount and rate oftranslational diffusion in an out of a responsive polymer membrane 202and within a proximity of magnetic particle(s) 201. Magnetic particle201 is capable of dephasing spins of solvent molecule(s) 203 withinresponsive polymer membrane 202 and dephasing is measurable using NMRdetection of NMR relaxation rates (1/T1, 1/T2). The flux or changes influx of solvent molecule(s) 203, in and out of responsive membrane 202,subsequently in and out of a magnetic field, magnetic field gradient ofthe nanoparticle 201, is proportional to NMR relaxation rates (1/T1,1/T2) of a solvent molecule 203 with susceptibility-induced dephasing bymagnetic nanoparticle 201. One or more discrete, continuous,differential, or integral NMR measurements are used to quantify themagnitude or amount of stimulus 204.

In FIG. 2, a matrix takes the form of a responsive polymer membrane 202is configured with one or more binding moieties 205,206 as shown in FIG.2 a with sensitivity and specificity to a physical or chemical stimulus204. When stimulus 204 is applied to device 200, the cross-linking ofresponsive polymer membrane 202 through one or more covalent bonds,hydrogen bonds, van der Waals interactions, or physical entanglement ofpolymer chains represented by element 207 are changed in a manner shownin FIG. 2 b. FIG. 2 b is a magnified representation of responsivepolymer membrane 209 which has transitioned from responsive polymermembrane 202 of FIG. 2 a upon receiving stimulus 204. Stimulus 204causes element 207 to transition to element 208 whereby an interactionbetween binding moiety 205 and binding moiety 206 reduces the extent ofcross-linking thus transforming polymer membrane 202 of FIG. 2 a intoresponsive polymer membrane 209. Responsive polymer 209 is morepermeable to solvent molecule(s) 203 thus enhancing the ability forsolvent molecule(s) 203 to diffuse in and out of responsive polymer 209and similarly in and out of magnetic field(s), magnetic fieldgradient(s) of magnetic particle(s) 201. The characteristics ofresponsive polymer membrane 202 of device 200 are reversible uponcessation of a stimulus 204. In the absence of stimulus 204, bindingmoiety 205 may re-establish cross-linking with binding moiety 206causing exclusion of solvent molecule(s) 203 from within a magneticfield(s), a magnetic field gradient(s) of a magnetic particle(s) 201 andsubsequently from a responsive polymer membrane. Magnetic particle(s)201 is (are) capable of dephasing spins of lesser proportion of solventmolecule(s) 203 within responsive polymer membrane 209 and dephasing ismeasurable using NMR detection of NMR relaxation rates (e.g., 1/T1,1/T2). The magnitude or amount of stimulus 204 is proportional to NMRrelaxation rates (1/T1, 1/T2) or delta of NMR relaxation rates ofsolvent molecules(s) 203. Thus device 201 is a sensor or transducer forstimulus 204 and may also be used as a flux sensor or flux transducer ofsolute, or solvent, or solution, or combination thereof. The magnitude,or amount, or rate of change of a stimulus 204 is quantified byproportion using one or more discrete, continuous, differential, orintegral measurements of NMR relaxation rates (e.g., 1/T1, 1/T2) with anNMR detector. Similarly, the magnitude, or amount, or rate of change ofamount of solvent(s) 203 flowing through responsive membrane 201 isquantified by proportion using one or more discrete, continuous,differential, or integral measurements of NMR relaxation rates (e.g.,1/T1, 1/T2) with an NMR detector. Device 200 may be calibrated formeasurement of stimulus 204 by collecting a data set of NMR relaxationrates proportional to a predetermined range or standard of magnitude oramount of stimulus 204 and determining one or more calibration factors.One or more calibration factors are then used to relate NMR relaxationrates to specific values for a stimulus 204. Similarly, device 200 maybe calibrated for measurement of magnitude, or amount, or rate of changeof an amount of solvent(s) 203 flowing through responsive membrane 201by collecting a data set of NMR relaxation rates proportional to apredetermined range or standard of magnitude, or amount, or rate ofchange of an amount of solvent(s) 203 flowing through responsivemembrane 201 and determining one or more calibration factors. One ormore calibration factors are then used to relate NMR relaxation rates tospecific values for magnitude, or amount, or rate of change of an amountof solvent(s) 203 flowing through responsive membrane 201.

FIG. 3 shows device 300 which is a magnetic particle 301 coated with aresponsive polymer matrix 302. A polymer matrix 302 is permeable tosolvent molecule(s) 303. An example of solvent molecule 303 is water.Responsive polymer matrix 302 contains cross-linked polymers.

FIG. 3 a is a magnified representation of responsive polymer matrix 302in one state. Responsive polymer matrix 302 can have one or more bindingmoiety(ies). FIG. 3 a shows a responsive polymer matrix 302 with bindingmoiety 305. Binding moiety 305 may be, e.g., an antibody or antigenattached to one or more polymer(s) of a responsive polymer matrix 302.Binding moiety 305 may also be, e.g., a nucleic acid polymer chain boundto one or more polymer chains of a responsive polymer matrix 302.Binding moiety 305 is attached to responsive matrix 302 through one ormore covalent bonds, hydrogen bonds, van der Waals interactions, orphysical entanglement with polymer chains. Responsive polymer 302 can bea hydrogel. The extent of cross-linking of responsive polymer matrix 302will change a specific volume fraction of device 300 leading to a changein diffusion time of solvent molecules 303 in proximity of magneticparticle 301. Magnetic particle 301 is capable of dephasing spins ofsolvent molecule(s) 303 and dephasing is measurable using NMR detectionof NMR relaxation rates (1/T1, 1/T2). A specific volume change of device300 and a change in diffusion time of solvent molecule(s) 303 isproportional to NMR relaxation rates (e.g., 1/T1, 1/T2) of a solventmolecule 303 with susceptibility-induced dephasing by magneticnanoparticle 301. One or more discrete, continuous, differential, orintegral NMR measurements are used to quantify magnitude or amount ofantigen or antibody within a solution or biological fluid sample.

In FIG. 3, in one embodiment, a responsive polymer matrix 302 isconfigured with a binding moiety 305 as shown in FIG. 3 a withsensitivity and specificity to an antigen 304. When a solutioncontaining the presence of antigen 304 is applied to device 300,cross-linking of responsive polymer matrix 302 through one or morecovalent bonds, hydrogen bonds, van der Waals interactions, or physicalentanglement of polymer chains changes in a manner shown in FIG. 3 b.FIG. 3 b is a magnified representation of responsive polymer 302 of FIG.3 a which has transitioned into responsive polymer matrix 306 uponinclusion of antigen 304. Antigen 304 causes responsive polymer matrix306 to increase the extent of cross-linking thus transforming polymermatrix 302 into responsive polymer 306. Responsive polymer 306 is lesspermeable to solvent molecule(s) 303 thus reducing the ability forsolvent molecule(s) 303 to diffuse in and out of responsive polymer 306and similarly in and out of a magnetic field, a magnetic field gradientof magnetic particle 301. A responsive polymer of device 300 can bereversible upon displacement of antigen 304. Displacement of antigen 304may take place by physical or chemical stimuli. In the absence ofantigen 304, responsive polymer 306 may transition back to responsivepolymer 302 enhancing flux of solvent molecule(s) 303 into a responsivepolymer matrix and, subsequently, within a magnetic field, a magneticfield gradient of magnetic particle 301. Magnetic particle 301 iscapable of dephasing spins of higher proportion of solvent molecule(s)303 within responsive polymer matrix 306 and dephasing is measurableusing NMR detection of NMR relaxation rates (e.g., 1/T1, 1/T2). Theamount or concentration of antigen 304 is proportional to NMR relaxationrates (e.g., 1/T1, 1/T2) or delta of NMR relaxation rates (e.g., Δ1/T1,Δ1/T2) of solvent molecules(s) 303. Thus device 301 is a sensor ortransducer for antigen 304. The amount, concentration, or rate of changeof concentration of antigen 304 is quantified by proportion using one ormore discrete, continuous, differential, or integral measurements of NMRrelaxation rates (e.g., 1/T1, 1/T2) with an NMR detector. Device 300 maybe calibrated for measurement of antigen 304 by collecting a data set ofNMR relaxation rates proportional to a predetermined range or standardof magnitude, amount, or concentration of antigen 404 and determiningone or more calibration factors. One or more calibration factors canthen be used to relate NMR relaxation rates to specific values formagnitude, amount, or concentration of antigen 404.

FIG. 4 shows device 400 which is a magnetic particle 401 coated with aresponsive polymer matrix 402. A polymer matrix 402 is permeable tosolvent molecule(s) 403. An example of solvent molecule 403 is watercontaining water protons. A responsive polymer matrix 402 containscross-linked polymers.

FIG. 4 a is a magnified representation of responsive polymer matrix 402in one state. Responsive polymer matrix 402 can have one or more bindingmoiety(ies). FIG. 4 a shows a responsive polymer matrix 402 with bindingmoiety 405 cross-linked with binding moiety 406. Binding moiety 405 andbinding 406 is antibody-antigen complex with each moiety bound to one ormore polymer chains of responsive polymer matrix 402. Binding moiety 405and binding moiety 406 enhances cross-linking of responsive polymermatrix 402 through their complexation. The extent of cross-linking bybinding moiety 405 and binding moiety 405 defines the physicalcharacteristics of responsive polymer matrix 402 including porosity,swelling, de-swelling, volume fraction, and permeability. Responsivepolymer 402 can be a hydrogel. The extent of cross-linking of responsivepolymer matrix 402 changes the specific volume fraction of device 400leading to a change in diffusion time of solvent molecules 403 in theproximity of magnetic particle 401. Magnetic particle 401 is capable ofdephasing spins of solvent molecule(s) 403 and dephasing is measurableusing NMR detection of NMR relaxation rates (e.g., 1/T1, 1/T2). Thespecific volume change of device 100 and change in diffusion time ofsolvent molecule(s) 403 is proportional to NMR relaxation rates (e.g.,1/T1, 1/T2) of a solvent molecule 403 with susceptibility-induceddephasing by magnetic nanoparticle 401. One or more discrete,continuous, differential, or integral NMR measurements are used toquantify amount, concentration, or rate of change of concentration ofantigen, antibody, or nucleic acid polymer 404.

In FIG. 4, for one embodiment, a responsive polymer matrix 402 isconfigured with antibody-antigen complex binding moieties 405,406 asshown in FIG. 4 a with sensitivity and specificity to each other. Whenfree antigen 404 is applied to device 400, the cross-linking ofresponsive polymer matrix 402 through antibody-antigen complex ofbinding moieties 105,106 are changed in a manner shown in FIG. 4 b. FIG.4 b is a magnified representation of responsive polymer 402 which hastransitioned into responsive polymer matrix 409 with the presence offree antigen 404. Free antigen 404 competes with one bound moiety of anantibody-antigen complex 405,406 and reduces the extent of cross-linkingof a responsive polymer matrix 402 between an antibody-antigen complex405,406 thus transforming polymer matrix 402 into responsive polymer409. Responsive polymer 409 is more permeable to solvent molecule(s) 403thus enhancing the ability for solvent molecule(s) 403 to diffuse in andout of responsive polymer 409 and similarly in and out of a magneticfield, a magnetic field gradient of magnetic particle 401. Responsivepolymer of device 400 can be reversible upon the absence of free antigen404. In the absence of free antigen 404, binding moiety 405 mayre-establish binding with binding moiety 406 causing exclusion ofsolvent molecule(s) 403 from within a magnetic field, a magnetic fieldgradient of the magnetic particle 401 and subsequently from responsivepolymer matrix 409. Magnetic particle 401 is capable of dephasing spinsof lesser proportion of solvent molecule(s) 403 within responsivepolymer matrix 409 and dephasing is measurable using NMR detection ofNMR relaxation rates (e.g., 1/T1, 1/T2). The magnitude or amount ofstimulus 404 is proportional to the NMR relaxation rates (e.g., 1/T1,1/T2) or delta of NMR relaxation rates (e.g., Δ1/T1, Δ1/T2) of solventmolecules(s) 403. Thus device 400 is a sensor or transducer for freeantigen 404. The amount, concentration, or rate of change ofconcentration of free antigen 404 can be quantified by proportion usingone or more discrete, continuous, differential, or integralmeasurement(s) of NMR relaxation rates (e.g., 1/T1, 1/T2) of solventmolecules(s) 403 with an NMR detector. Device 400 may be calibrated formeasurement of free antigen 404 by collecting a data set of NMRrelaxation rates proportional to a predetermined range or standard ofmagnitude, amount, or concentration of free antigen 404 and determiningone or more calibration factors. One or more calibration factors canthen be used to relate NMR relaxation rates to specific values formagnitude, amount, and/or concentration of free antigen 404.

FIG. 5 shows device 500 which is a magnetic particle 501 coated with aresponsive polymer matrix 502. A polymer matrix 502 is permeable tosolvent molecule(s) 103. An example of solvent molecule 503 is watercontaining water protons. Responsive polymer matrix 502 containscross-linked polymers.

FIG. 5 a is a magnified representation of responsive polymer matrix 502in one state. Responsive polymer matrix 502 can have one or more bindingmoiety(ies). FIG. 5 a shows responsive polymer matrix 502 with one ormore binding moiety(s) 505 cross-linked with binding moiety 106 havingone or more binding sites with affinity for binding moiety 505. Bindingmoiety 505 and binding moiety 506 are cross-linked through one or morehydrogen bonds, van der Waals interactions, or physical entanglement.The extent of cross-linking between one or more binding moiety 505 andbinding moiety 506 defines physical characteristics of a responsivepolymer matrix 502 including porosity, swelling, de-swelling, volumefraction, and permeability. Responsive polymer 502 can be a hydrogel.The extent of cross-linking of responsive polymer matrix 502 changes thespecific volume fraction of device 500 leading to a change in diffusiontime of solvent molecules 503 in the proximity of magnetic particle 501.Magnetic particle 501 is capable of dephasing spins of solventmolecule(s) 503 and dephasing is measurable using NMR detection of NMRrelaxation rates (e.g., 1/T1, 1/T2). The specific volume change ofdevice 500 and the change in diffusion time of solvent molecule(s) 503is proportional to NMR relaxation rates (e.g., 1/T1, 1/T2) of a solventmolecule 503 with susceptibility-induced dephasing by magneticnanoparticle 501. One or more discrete, continuous, differential, orintegral NMR measurements are used to quantify amount, concentration, orrate of change of concentration of analyte 504.

In FIG. 5, a responsive polymer matrix 502 is configured with one ormore binding moieties 505,506 as shown in FIG. 5 a with sensitivity andspecificity to an analyte 504. In one embodiment, analyte 504 hassimilar or equivalent reactive group(s) to binding moiety 505 so ananalyte 504 of interest or its equivalent is attached to responsivepolymer matrix 502 in advance. When free analyte 504 is applied todevice 500, the cross-linking of responsive polymer matrix 502 ischanged in a manner shown in FIG. 5 b. FIG. 5 b is a magnifiedrepresentation of responsive polymer 502 which has transitioned intoresponsive polymer matrix 509 in the presence of free analyte 504. Freeanalyte 504 competes with one or more bound moiety(ies) of complex505,506 and displaces bound moiety 506 from cross-linking withresponsive polymer matrix 502, thus transforming polymer matrix 502 intoresponsive polymer matrix 509. Responsive polymer 509 is more permeableto solvent molecule(s) 503, thus enhancing the ability for solventmolecule(s) 503 to diffuse in and out of responsive polymer 509 andsimilarly in and out of a magnetic field, a magnetic field gradient of amagnetic particle 501. A responsive polymer of device 500 can bereversible in the absence of analyte 504. In the absence of analyte 504,binding moiety 505 may re-establish cross-linking with binding moiety506 causing exclusion of solvent molecule(s) 503 from within a magneticfield, a magnetic field gradient of a magnetic particle 501 andsubsequently from responsive polymer matrix 509. Magnetic particle 501is capable of dephasing spins of lesser proportion of solventmolecule(s) 503 within responsive polymer matrix 509 and dephasing ismeasurable using NMR detection of NMR relaxation rates (e.g., 1/T1,1/T2). The magnitude or amount of stimulus 504 is proportional to theNMR relaxation rates (e.g., 1/T1, 1T/2) or delta of NMR relaxation rates(e.g., Δ1/T1, Δ1/T2) of solvent molecules(s) 503. Thus device 500 is asensor or transducer for analyte 504. The amount, concentration, or rateof change of concentration of analyte 504 can be quantified byproportion using one or more discrete, continuous, differential, orintegral measurement(s) of the NMR relaxation rates (e.g., 1/T1, 1T/2)of solvent molecules(s) 503 with an NMR detector. Device 500 may becalibrated for measurement of free analyte 504 by collecting a data setof NMR relaxation rates proportional to a predetermined range orstandard of magnitude, amount, or concentration of free analyte 504 anddetermining one or more calibration factors. One or more calibrationfactors are then used to relate NMR relaxation rates to specific valuesfor magnitude, amount, or concentration of free analyte 504.

In one embodiment, devices 100 of FIG. 1 are prepared using asurfactant-free emulsion polymerization (SFEP) to encapsulate each ironoxide particle of approximately 10 nm in diameter within a largespherical responsive polymer matrix 102. This will produce a stable,compact and chemically stable polymer overlay. Before emulsionpolymerization, surfaces of each iron oxide particle may be modified byadsorption of oleic acid. In one method, a jacketed cylindrical reactionvessel was changed with 180 mL of water and 20 mL of the iron oxidedispersion. After 30 minutes of deoxygenating, 30 mL of styrene (St.Aldrich), 3 mL of methyl methacrylate (MMA, Aldrich), and 0.2 g sodiumstyrene sulfonate (NaSS, Polyscience) were added into the vessel. Thetemperature was increased to 70° C. and 2.0 g ammonium persulfate (APS,Aldrich) was added to initiate polymerization, which reacted for 5hours. In another method, after adsorption of oleic acid, anapproximately 94:6 wt % ratio of poly (N-isopropylacrylamide)—NIPAM(26.1 mL, 0.01 M): acrylic acid—Aac (1.6 mL, 0.01 M) is then added andstirred. The solution is heated to 71° C. in an oil bath, and then APS(0.8 mL, 0.01 M) added to initiate the polymerization. The reactiontime, which depends on the amount of starting materials, is variedbetween 6 and 8 hours. At the end of this period, the solution is cooledand filtered through a 1 micron membrane to remove any micrometer sizeimpurities and or any aggregate particles. The filtered solution may becentrifuged at 20° C. for 2 hours at 3500 rpm and the supernatantseparated to remove unreacted materials, soluble side products, andseeds of pure polymer. The purified nanoparticles are than diluted withpure water and stored at room temperature. The size of the hydrogelcoated nanoparticles can be varied between 100-230 nm by controlling theamount of monomer and initiator as well as reaction time. Specificbinding moieties may be attached to the hydrogel with using knowncovalent conjugation and or physical adsorption methods.

FIG. 6 is a schematic diagram 600 of an NMR system for detection of anecho response of a sample 603 to an RF excitation, thereby detecting thepresence and/or concentration of an analyte in a sample. In a specificembodiment, bias magnets 601 establish a bias magnetic field Bb 602through a sample 603. In alternative embodiments, other configurationsof bias magnetic field Bb 602 can be applied to sample container 604including, unilateral magnetic fields, low powered magnetic fields, andthe earth's magnetic field. Reagents (e.g., including, e.g., providednanosensors) 608 are added in sample container 604 prior or introducedsimultaneously with sample 603 into container 604. An RF coil 605 and RFoscillator 606 provides an RF excitation at the Larmor frequency whichis a linear function of the bias magnetic field Bb. In one embodiment,RF coil 605 is wrapped around sample container 604. In alternativeembodiments, RF coil 605 can be a planar RF coil or other shape and formof RF coil can be used with sample container 604. The excitation RFcreates instability in the spin of water protons (or free protons in anon-aqueous solvent) within responsive hydrogel matrices of nanosensors.When the RF excitation is turned off, protons “relax” to their originalstate and emit an RF signal characteristic of the concentration ofanalyte. Coil 605 acts as an RF antenna and detects an “echo” of therelaxation. The echoes of interest are decay in time T1 and/or T2. TheRF signal from coil 605 is amplified by amplifier 607 and processed todetermine a change in decay time (e.g., T1, T2) in response toexcitation in the bias field Bb 602. Various configurations of container604 (e.g., well, channel, reservoir, etc.) may be used for analytedetection.

FIG. 7 is a schematic diagram 700 of an NMR system for detection of anecho response of a solution 703 to an RF excitation, thereby detecting aphysical or chemical stimulus 709 applied to solution 703 containing thenanosensors 708 (e.g., provided nanosensors). In a specific embodiment,bias magnets 701 establish a bias magnetic field Bb 702 through solution703. In alternative embodiments, other configurations of bias magneticfield Bb 702 can be applied to structure 704 including, unilateralmagnetic fields, low powered magnetic fields, and the earth's magneticfield. Provided nanosensors 708 are contained within structure 704.Structure 704 may be an open or sealed container including but limitedto a well, a glass capillary, a glass cell, or any structure forconfining a volume of solution containing one or more nanosensors 708.Structure 704 can be made of any material that can amplify, enhance, orretard, in a controlled manner, the magnitude of the physical orchemical stimulus applied to solution 703. Structure 704 may bepermeable to a physical or chemical stimulus applied to solution 703.Various transducers, conductors, resistors, can be applied to one ormore surface(s) or incorporated within one or more surface(s) ofstructure 704 including electrodes, piezoelectric materials, andthermocouples. An RF coil 705 and RF oscillator 706 provides an RFexcitation at the Larmor frequency which is a linear function of thebias magnetic field Bb 702. In one embodiment, RF coil 705 is wrappedaround structure 704. In alternative embodiments, RF coil 705 can be aplanar RF coil or other RF coil shape and form of can be used withstructure 704. The excitation RF creates instability in the spin ofwater protons (or free protons in a non-aqueous solvent) withinresponsive hydrogel matrices of nanosensors 708. When the RF excitationis turned off, protons “relax” to their original state and emit an RFsignal characteristic of a physical or chemical stimulus applied tosolution 703. Coil 705 acts as an RF antenna and detects an “echo” ofthe relaxation. The echoes of interest are decay in time (e.g., T1and/or T2). The RF signal from the coil 705 is amplified 707 andprocessed to determine a change in decay time (e.g., T1, T2) in responseto excitation in the bias field Bb 702. Sample solution 703 may bereplaceable with a hydrogel or a membrane containing one or morenanosensors 708.

FIG. 8 is a schematic diagram 800 of an NMR system for detection of anecho response of a layer 803 containing one or more nanosensors (e.g.,provided nanosensors) attached or preferably coated onto one or morewall of device implant 804. Layer 803 can contain discrete nanosensorsattached by nonspecific absorption or specific chemical coupling to atleast one surface of device implant 804. Nanosensors may be partiallycoated with a responsive coating whereby the coated portion ofnanosensors excludes portions of nanoparticles attached to a surface ofdevice implant 804. Device implant 804 may be placed into one or morebiologic cell, organ, or whole body 808. In a specific embodiment, biasmagnets 801 establish a bias magnetic field Bb 802 to layer 803 ofdevice implant 804 within body 808. In alternative embodiments, otherconfigurations of bias magnetic field Bb 802 can be applied to abiologic body 808 including, unilateral magnetic fields, low poweredmagnetic fields, and the earth's magnetic field. An RF coil 805 and RFoscillator 806 provides an RF excitation at the Larmor frequency whichis a linear function of the bias magnetic field Bb 802. In oneembodiment, RF coil 805 is wrapped around body 808. In alternativeembodiments, RF coil 805 can be a planar RF coil or other RF coil shapeand form of can be used to apply an RF excitation to body 808 andsubsequently to layer 803. The excitation RF creates instability in thespin of water protons (or free protons in a non-aqueous solvent) withinresponsive hydrogel matrices of nanosensors of layer 803. When the RFexcitation is turned off, the protons “relax” to their original stateand emit an RF signal characteristic of the physical parameters sensedby layer 803. Coil 805 acts as an RF antenna and detects an “echo” ofthe relaxation. The echoes of interest are the decay in time (e.g., T1and T2). The RF signal from coil 805 is amplified 807 and processed todetermine a change in decay time (e.g., ΔT1, ΔT2) in response to theexcitation in the bias field Bb 802. In an alternative embodiment,device implant 804 coated with layer 803 can be monitored using an NMRimager.

FIG. 9 is a cartoon schematic of a principle of operation and elementsof a smart device 900. Smart device 900 comprises a matrix 901 having asensing element 902 and a control release element 903. Smart device 900can be placed within, inserted into, or implanted inside a biologic,cell, organ, or body 904. Sensing element 902 can be one or moreprovided transducers. Sensing element 902 may emit one or more RF echosignal 906 generated using the NMR principle and methods of detection asdescribed herein. One or more RF echo signal 906 may be detected by a RFantenna 905 of an NMR detector. Sensing element 902 providesmeasurements of one or more physical, chemical or analyte stimuli withinan environment and in the vicinity of smart device 900. Measurementresults from sensing 902 can be used to control a physical or chemicalstimulus emitter 907. Stimulus emitter 907 can be used to apply astimulus 908 to control release element 903. Reception of stimulus 908by control release element 903 allows one or more of the followingbinding moieties: an atom, an ion, a molecule, a compound, a catalyst,an enzyme, an electroactive mediator, an electron-pair donor, anelectron-pair acceptor, a lanthanide, an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent, a biological molecule, ametabolite of a therapeutic agent, a peptide, a polypeptide, a protein,a carbohydrate, a polysaccharide to be released or delivered out ofmatrix 901 and into a biologic, cell, organ, or body 904. Smart device900 can be configured to operate in an open loop, as previouslydescribed, or closed-loop control manner. In a closed-loop manner, smartdevice 900 may comprise a sensing element 902, a miniaturized NMRdetector functioning in conjunction with sensing element 902, a controlrelease element 903, a stimulus emitter 907, microelectronics forimplementing a close-loop control system, and a power source. As anexample, element 902 can detect glucose and control release element 903is capable of releasing insulin. Element 902 can glucose concentrationwithin body 904 and will release insulin in a controlled manner as toallow body 904 to establish or maintain a normal glucose concentration.Smart device 900 will thus function as an artificial pancreas.

Provided NMR detection systems can measure either a positive or negativechange in relaxation rates (e.g., ΔT1 and ΔT2). In one embodiment,provided systems and methods measure T2 changes due to a stimulus or ananalyte binding event, leading to positive and negative T1 or T2changes. In one aspect, an NMR detection system will measure baseline T2of a solution containing a responsive polymer coated nanoparticlesfirst, and then will be mixed with liquid sample containing an analyte,and T2 will be measured again to determine whether a change in T2 hasoccurred in the presence of analyte. A quality control step can beperformed to minimize errors that may affect the measurement of analyteincluding stoichiometry, metering, mixing, variations in nanoparticleproperties, and fluidic transport.

In one embodiment provided compositions and methods can be used todetect analyte by measuring NMR echo signals from a sample at a singletime. Alternatively, an NMR detection system can perform a series ofmeasurements spanning a period of time and/or may compare or analyzemeasurements to improve detection of analyte. For example, bindingbetween analyte and nanosensors may proceed during an interval which islonger than the time required for a particular measurement. An NMRsystem may perform measurements repeatedly to observe changes caused bybinding. Repeated measurements can greatly enhance accuracy of resultsby reducing false negatives and/or false positives, providing a lowerdetection threshold, and enhancing the detection probability for a givenstimulus or quantity of an analyte. An NMR detection system can alsoderive parameters related to reaction kinetics from repeatedmeasurements on a same sample, including a rate of change of a parameteror an accumulated reaction parameter. A multiple scanning protocolcombined with a rate-magnitude analysis can enhance both reliability andthreshold sensitivity of a detection system.

A model suggests a positive T2 change is due to reactions whereby watermolecules are displaced further from a responsive polymer coatednanoparticles upon an occurrence of a stimulus or analyte binding event.A negative T2 change is due to repeated dephasing of more watermolecules diffusing in closer proximity to nanoparticles upon a stimulusor analyte binding event. A simplified nanoparticle is assumed toconsist of a spherical core of superparamagnetic material, surrounded bya spherical shell of hydrogel, all in water. However, the model can beapplied or modified for use with nanoparticles of other shapes and foruse with other solvents. Without being bound by theory, the modelsuggests the following mechanisms for observed T2 changes:

(1) Nanoparticles in solution reduce T2 relative to plain water. Themodel suggests that depolarization is due to a dipole magnetic fieldproduced by a magnetized core. Field distortion causes spins to precessat different frequencies, leading to destructive interference. AlthoughCPMG normally refocuses static field-nonuniformity effects, Brownianmotion of water molecules causes them to enter and exit fielddistortions in a time shorter than the echo interval, thereby making thespin dispersion time-dependent and breaking the CPMG refocusing effect.

(2) Water flux in the proximity of nanoparticles result in delta changesof T2. Delta changes in T2 are directly proportional to specific volumefraction of responsive polymer coated nanoparticles and diffusion ofwater in the proximity of the nanoparticles. Water flux within thespherical shell are due to analyte molecules modifying permeability of aspherical shell surrounding nanoparticles and regions around ananoparticle, thereby enhancing or decreasing water fluxes from thatregion, changing the specific volume fraction of the spherical shell. Achange in specific volume fraction effects the diffusion interactionbetween nanoparticles and water molecules thus enhancing or reducingspin dispersion, and increasing or decreasing T2. A single exponentialusually fits the polarization decay curve of T2. In the case of a T2decrease, hydrogens close to nanoparticles are strongly dephased, whilea general solvent sees only a uniform field, a two-population system.However, spin populations are rapidly equilibrated across a sample byspin diffusion via homonuclear flip-flop interactions, resulting in asingle averaged T2.

Provided NMR detection systems may detect a stimulus or presence of ananalyte by analyzing the magnetic resonance signals by spectral analysisto seek a frequency component characteristic of the occurrence of astimulus or analyte binding event. Alternatively the step could includeapplying a CPMG procedure, and analyzing signals to determine T1 and/orT2 of a solution. The T1 or T2 distribution may be a single exponentialcomponent, or it may include a multitude of components, depending onspin diffusion rate. A delta T1 or delta T2 from a baseline measurementof nanosensors without a stimulus or analyte and indicates theoccurrence of a stimulus or presence of an analyte.

Provided NMR detection measurement methods may include the steps ofmeasuring T1 or T2 value of a standard. Here a standard is any materialwhich has a known T1 or T2. Preferably the T1 or T2 of the standard isunchanging in time and is known from prior calibration measurements. Forexample a standard may be a solution of nanosensors with a concentrationadjusted to provide a particular value of T1 or T2. Standards enabledetection and correction of instrumentation drifts. A standard may be asolution or encapsulation of nanosensors selected to have a T1 or T2 ina desired range. A standard may be arranged to have a T1 or T2substantially equal to that of a stimulus-free or an analyte-freesample; an analytical negative control. The standard may have a T1 or T2close to that produced by a stimulus or analyte; a analytical positivecontrol. The method may include measuring T1 or T2 of multiple standardswith different T1 and T2 values.

In an illustrative, constructive embodiment, device 100 of FIG. 1 isdesigned as a temperature nanosensor. Binding moieties 105, 106comprises hydrophobic groups such as methyl, ethyl and propyl groups.For example, responsive polymer matrix 102, 109 is poly(N-isopropylacrylamide) (PNIPAAm). PNIPAAm has a low critical solutiontemperature (LCST) in the range of 25-32° C. PNIPAAm decreases theirwater-solubility as temperature increases and shrink as temperatureincreases above the LCST. At low temperatures, hydrogen bonding betweenhydrophilic segments of binding moieties 105, 106 and water dominates,leading to enhanced dissolution of water. As temperature increases,hydrophobic interactions among hydrophobic segments of binding moieties105, 106 become strengthened, while hydrogen bonding becomes weaker. Thenet result is shrinking of responsive matrix 102, 109 due tointer-polymer chain association through hydrophobic interactionsresulting in flux of water of out of responsive matrix 102,109. Adecrease in specific volume fraction of matrix 102 will result in adecreased NMR relaxation rate (e.g., 1/T1, 1T/2). In this design,temperature is inversely proportional to NMR relaxation rates.

In an illustrative, constructive embodiment, device 200 of FIG. 2 isdesigned as an electro-sensitive hydrogel membrane. Binding moieties205, 206 of one or more nanosensors within a hydrogel membrane or amembrane is made of sodium acrylic acid-acrylamide copolymer. Membranedevice 200 contains an aqueous solution (e.g., acetone and water).Membrane device 200 is placed between two planar electrodes. The planarelectrodes are used to apply and electric field to a solution containingmembrane device 200. In the absence of electrolytes or in the presenceof low concentration of electrolytes, application of an electric fieldwill cause the membrane and/or one or more nanosensors to shrink. Forexample, due to a migration of sodium ion Na⁺ to a cathode electrodewill result in changes in carboxyl groups of polymer chains one or morebinding moiety 205, 206 from —COO⁻Na⁺ to —COOH. In the presence of highconcentration of electrolytes in solution, however, more Na⁺ enters themembrane and the membrane swells. Shrinking and swelling due to theapplied electric field decreases and increases specific volume fractionof the membrane resulting in decreases and increases of NMR relaxationrates (e.g., 1/T1, 1T/2). NMR echo signals may be used to detectelectrolyte concentration and/or their migration in a solution.

In an illustrative, constructive embodiment, device 100 of FIG. 1 isdesigned as a glucose nanosensor. Responsive matrix 102 is made of ablock co-polymer of N,N dimethylaminoethyl methacrylate (DEA) and2-hydroxypropyl methacrylate (HPMA) with cross-linking binding moieties105,106 comprising glucose oxidase immobilized by polyacrylamide polymerchains. In the presence of glucose, glucose oxidase converts glucose toproduce gluconic acid which changes the pH of the environment withinresponsive matrix 102. Responsive matrix 102 swells as a result from theionization of the amine groups by the lowering pH. An increase inspecific volume fraction of responsive matrix 102 results in a decreasethe NMR relaxation rates (e.g., 1/T1, 1T/2). In this design, glucoseconcentration is inversely proportional to NMR relaxation rates.

In an illustrative, constructive embodiment, device 400 of FIG. 4 isdesigned as an analyte specific nanosensor. An antigen-antibody complexusing binding moieties 405, 406 is incorporated into responsive matrix402. For example, rabbit immunoglobulin G (IgG), the antigen, ischemically modified by coupling it with N-succinimidylacrylate (NSA) inphosphate buffer solution to introduce vinyl groups into the rabbit IgG.Resultant vinyl rabbit IgG is then mixed with antibody, goat rabbit IgG(GAR IgG), to form an antigen-antibody complex. The vinyl-rabbit IgG isthen co-polymerized with acrylamide (AAm) as a comonomer an N,Nmethylenebisacrylamide (MBAA) as a cross-linker in the presence of GARIgG, resulting in responsive matrix 402 containing antigen-antibody bondsites 405, 406. One or more device 400 is mixed into a buffer solutionwith rabbit IgG. In the presence of free rabbit IgG, theantigen-antibody entrapment hydrogel matrix 402 will swell in proportionto the concentration of free rabbit IgG. Free IgG induces disassociationof antigen-antibody bonds grafted to inside responsive polymer matrix402, due to the stronger affinity of antibody for the free antigen thanthe for antigen grafted to the network of polymer within responsivematrix 402. Therefore, responsive polymer matrix 402 swells in thepresence of the free antigen because of disassociation ofantigen-antibody bonds resulting in a decreased in cross linkingdensity. Swelling due to the presence of free antigen increases thespecific volume fraction of polymer matrix 402 resulting in an increaseof the NMR relaxation rates (e.g., 1/T1, 1T/2). The NMR echo signals maybe used to detect amount or concentration of free antigen. For example,free antigen concentration is inversely proportional to NMR relaxationrate (e.g., 1/T2).

In an illustrative, constructive embodiment, device 500 of FIG. 5 isdesigned as a glucose nanosensor. Responsive matrix 502 comprises poly(2-glucosyloxyethel methacrylate) (PGEMA) with binding moieties 505,pendant glucose groups, having affinity for binding moiety 506 which isCon A. Device 500 in an aqueous solution is flocculated by the additionof Con A through the complex formation between Con A and the pendantglucose groups of PGEMA. In the he presence of free glucose hydrogelmatrix 502 will swell in proportion to the concentration of freeglucose. Free glucose induces disassociation of the complex formationbetween Con A and pendant glucose groups of PGEMA inside responsivepolymer matrix 502, due to the stronger affinity of the Con A for freeglucose than pendant glucose groups of PGEMA grafted to the network ofpolymer within responsive matrix 502. Therefore, responsive polymermatrix 502 swells in the presence of the free glucose because ofdisassociation of the complexation bonds between Con A and pendantglucose groups of PGEMA resulting in a decreased in cross-linkingdensity. Swelling due to the presence of free glucose increases thespecific volume fraction of responsive 502, resulting in an increase ofNMR relaxation rates (e.g., 1/T1, 1T/2). NMR echo signals may be used todetect amount or concentration of free glucose. For example, freeglucose concentration is inversely proportional to NMR relaxation rate(e.g., 1/T2).

In FIG. 10 and FIG. 11, a principle on which the invention is based isexplained on the basis of one embodiment, and serves not to limit theinvention to a particular embodiment but for the purpose of explainingthe principle. FIG. 10A shows the theoretical shift in T2 obtained byincreasing size of a nanosensor. As particle diameter increases, T2decreases in response to a certain point of diminishing return, wherethe T2 measurement begins to flatten out then reverses and increaseswith further increases in particle size. Provided methods compriseaddition of a binding agent to a single population of analyte-boundnanoparticles. The method involves coating a nanoparticle with a singlelayer of analyte, and a single layer of binding agent. Addition ofbinding agent in this assay format leads to a uniform increase in sizeamong all nanoparticles. Sze increase is dependent on concentration ofbinding agent and size of the binding agent and analyte, which are eachhighly adjustable. We hypothesize that this layer formation isthermodynamically and sterically favorable over cluster formation.Further, provided methods result in formation (or dispersion) and assayof a single population of non-aggregated nanosensors, thereby allowingfor optimized and accurate measurements of relaxation parameters.

Provided methods comprise competitive assays which take advantage ofthese benefits. Methods comprise competitive assays consisting ofinhibitive competitive assays, wherein formation of nanosensor coatingis competitive, as well as dispersive competitive assays, whereindispersion of pre-formed nanosensor coating is competitive. FIG. 11Adepicts a schematic of one preferred format for a competitive coatingassay according to the provided methods. Analyte, optional analyteanalog, and binding agent (depicted in the scheme as antibody) areselected depending on the analyte desired for detection. Further, thetype, size, or modification of binding agent is selected to optimizeparticle size when a nanosensor is saturated, for optimal relaxationmeasurements, and detection of analyte. Exemplary reagent production anduses in detection assays are provided below.

Crosslinked Iron Oxide Particle Synthesis

Nanoparticles were prepared according to methods known in the art.Briefly, T-10 dextran was dissolved in water mixed with ferric chlorideand degassed by nitrogen purging. Ferric chloride solution was added tothe mixture and the pH brought to 10 with ammonium hydroxide. Resultingmonodisperse iron oxide (MION) particles were crosslinked withepichlorohydrin and ammonia to provide stability and amine groups forconjugation to targeting moieties to produce crosslinked iron oxide(CLIO) nanoparticles. Fluorescein decorated CLIOs were prepared byincubating aminated 30 nm CLIO nanoparticles with fluoresceinisothiocynate (FITC). Unreacted FITC was then removed by size exclusiongel filtration in columns packed with Sephadex G-25 beads. ConjugatedFITC-CLIO nanoparticles were characterized with an iron assay forparticle concentration as well as against a fluorescent standard curveto measure the number of fluorescein molecules per particle.

Optimization of Particle Size and Relaxation Measurement

Anti-fluorescein antibody coating assays were performed by titrating invarying amounts of antibody (either anti-FITC antibody or anti-FITCantibody conjugated to an 80 kDa alkaline phosphatase (AP) protein) into a fixed concentration of nanoparticles. Fluorescein decorated CLIOnanoparticles prepared as described above were incubated with bindingagent (antibody/conjugated antibody) for a desired length of time at 37°C. and then T₂ relaxation times were measured on a Bruker Minispec. Thecoating assay had several unique advantages over nanoparticle clusteringassays. The coating assay format was energetically favorable for thespecific nanoparticle/antibody combinations tested which allowed themeasured T2 switch to be robust and controllable. The recovery of theswitch in a competitive assay is therefore also straightforward. Asdiscussed above, and depicted in FIG. 10A, a decrease in measured T2relaxation time is enhanced when a binding agent size is increased(e.g., antibody tethered to a large molecule (e.g., a protein, antibody,conjugated antibody)), which effectively results in a thicker coataround a nanoparticle. FIG. 10B shows the experimental results ofcoating assays using anti-flourescein antibody or antibody conjugated toalkaline phosphatase. As more antibodies are titrated in to the reactionand more nanoparticles become saturated with antibody, the T2 decreasesin response. The curve shape was steeper with the anti-FITC-AP titrationindicating that the particles are coated with a larger coating thicknessas compared to the anti-FITC antibody case. Particle size measurements(using a Malvern Zetasizer) of nanosensors with increasing amounts ofantibody or AP-conjugated antibody confirm the size increases withincreasing antibody as well as further increases using conjugatedantibody, and corroborate the hypothesis that he particles are beingcoated with anti-FITC-antibodies and with anti-FITC-AP antibodies. Asantibody concentration was increased, the measured size of thenanoparticle increases proportionately. The T2 graph and sizemeasurements also demonstrated a limitation of the coating assay format.Further increasing the size of the coating with binding agent willenhance the T2 switch with diminishing returns until the T2 flattens outand then begins to increase with size as shown FIG. 10A. Once a particleis fully coated with antibody, further changes in T2 cannot be measuredunless the coating is disrupted.

Competitive Assays

Prepared nanoparticles were used in two different fluoresceincompetitive assay formats—inhibitive and competitive (dispersive).Inhibitive assays were performed by pre-incubating varyingconcentrations of fluorescein analyte with a fixed amount ofanti-fluorescein antibody prior to incubation with preparednanoparticle. For the competitive (dispersive) format, fluorescentanalyte was incubated with a prior prepared antibody-nanoparticlesolution. Both competitive formats demonstrated a positive correlationbetween measured T2 and analyte concentration as shown in FIG. 11A.

Inhibitive assays were performed in two-steps by pre-incubating varyingconcentrations of fluorescein analyte with a fixed amount ofanti-fluorescein antibody prior to incubation with nanoparticle. Forexample, 80 ul of 62.5 ug/ml anti-FITC-AP antibody was fixed with avarying concentration of fluorescein sodium salt (analyte). The solutionwas incubated for 30 min at 37°. After pre-incubation, 240 ul of 0.067mM [Fe] nanoparticles prepared as described above were added. Thecomplete solution was incubated for 15 min at 37° followed bymeasurement of T₂ relaxation times on a Bruker Minispec. Results areshown in FIG. 12A.

The competitive (dispersive) format involved incubating fluorescentanalyte in one step with a pre-incubated antibody-nanoparticle solution.For this assay, 3 concentrations of anti-FITC-AP antibody (125, 250 or500 ul/ml) were mixed with a fixed concentration of nanoparticles. Thesolution was incubated at 37° prior to adding varying concentrations offluorescein (analyte). The complete solution was incubated at 37°followed by measurement of T₂ relaxation times on a Bruker Minispec.Results are shown in FIG. 12B.

Equivalents

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A system for the detection of an analyte in a liquid sample, thesystem comprising at least one nanoparticle comprising a polymer matrixlayer about a magnetic core, wherein the polymer matrix layer has aspecific volume that varies as a function of a presence and/orconcentration of the analyte in the liquid sample, and wherein a changein the specific volume of the polymer matrix layer corresponds to achange in an NMR-measured property of protons in the liquid sample inthe vicinity of the at least one nanoparticle.
 2. The system of claim 1,wherein the NMR-measured property is a T₁ or T₂ relaxivity of protons of(substantially) freely diffusing water molecules in the liquid sample(e.g., wherein the protons are dephased upon RF excitation).
 3. Thesystem of claim 1, wherein the polymer matrix layer comprises ahydrophilic mesh comprising binding moieties responsive to the presenceand/or concentration of the analyte in the liquid sample.
 4. The systemof claim 1, wherein the liquid sample is a biological sample; whereinthe at least one nanoparticle and the liquid sample are in vivo; and/orwherein the at least one nanoparticle and the liquid sample are ex vivo.5. The system of claim 3, wherein the binding moieties mediate theextent of cross-linking of the matrix as a function of the presenceand/or concentration of the analyte, wherein the specific volume of thematrix is a function of the extent of cross-linking; and/or wherein thebinding moieties comprise at least one member selected from the groupconsisting of an amino group, a carboxyl group, a sulfhydryl group, anamine group, an imine group, an epoxy group, a hydroxyl group, a thiolgroup, an acrylate group, and an isocyano group.
 6. The system of claim3, wherein the analyte comprises at least one member selected from thegroup consisting of a protein, a peptide, a polypeptide, an amino acid,a nucleic acid, an oligonucleotide, a therapeutic agent, a metabolite ofa therapeutic agent, RNA, DNA, an antibody, an organism, a virus, abacteria, a carbohydrate, a polysaccharide, and glucose.
 7. The systemof claim 1, wherein the at least one nanoparticle is paramagnetic orsuperparamagnetic.
 8. The system of claim 1, the system comprising aplurality of said nanoparticles arranged in a network.
 9. The system ofclaim 8, wherein said nanoparticles are arranged in said network toallow substantially free diffusion of water molecules in the vicinity ofeach individual nanoparticle.
 10. The system of claim 8, wherein saidnanoparticles are at least partially immobilized on a surface (forexample, by nonspecific absorption, by specific chemical coupling, or byspecific binding); and/or wherein a part of each of said nanoparticlesis immobilized on said surface, and another part of said eachnanoparticle is exposed to said liquid sample; and/or wherein saidnanoparticles each comprise a core that is only partially coated withsaid polymer matrix layer.
 11. The system of claim 10, wherein saidnanoparticles comprise a partial polymer coating, wherein said partialpolymer coating of each of said nanoparticles is exposed to said liquidsample.
 12. A system for the detection of a property of a liquid sample,the system comprising at least one nanoparticle comprising a polymermatrix layer about a magnetic core, wherein the polymer matrix layer hasa specific volume that varies as a function of the liquid sampleproperty to be detected, and wherein a change in the specific volume ofthe polymer matrix layer corresponds to a change in an NMR-measuredproperty of protons in the liquid sample in the vicinity of the at leastone nanoparticle.
 13. The system of claim 12, wherein the detectedproperty of the liquid sample is a member selected from the groupconsisting of static pH, dynamic pH, and ionic strength; and/or whereinthe detected property of the liquid sample is a concentration of one ormore of the following in the liquid sample: a lipid, a gas (e.g.,oxygen, carbon dioxide), an electrolyte (e.g., sodium, potassium,chloride, bicarbonate, BUN, creatinine, glucose, magnesium, phosphate,calcium, ammonia, lactate), a lipoprotein, cholesterol, a fatty acid, aglycoprotein, a proteoglycan, and/or a lipopolysaccharide.
 14. A methodfor detecting an analyte in a liquid sample, the method comprising thesteps of: (a) exposing at least one magnetic nanoparticle to a liquidsample, wherein the at least one nanoparticle comprises a polymer matrixlayer about a magnetic core, the polymer matrix layer having a specificvolume that varies as a function of the presence and/or concentration ofthe analyte; and/or (b) applying an external magnetic field to the atleast one magnetic nanoparticle; (c) applying an RF excitation to detectprotons of freely diffusing water molecules in the liquid sample in thevicinity of the at least one magnetic nanoparticle; (d) measuring a T₁and/or T₂ relaxivity following application of the RF excitation; and (e)determining the presence and/or concentration of the analyte in theliquid sample using the T₁ and/or T₂ relaxivity.
 15. The method of claim14, further comprising the step of determining one or more calibrationcurves relating the T₁ and/or T₂ relaxivity to the presence and/orconcentration of the analyte in solution.
 16. A method for detecting aproperty of a liquid sample, the method comprising the steps of: (a)exposing at least one magnetic nanoparticle to a liquid sample, whereinthe at least one nanoparticle comprises a polymer matrix layer about amagnetic core, the polymer matrix layer having a specific volume thatvaries as a function of the liquid sample property to be detected; (b)applying an external magnetic field to the at least one magneticnanoparticle; (c) applying an RF excitation to detect protons of freelydiffusing water molecules in the liquid sample in the vicinity of the atleast one magnetic nanoparticle; (d) measuring a T₁ and/or T₂ relaxivityfollowing application of the RF excitation; (e) determining the liquidsample property using the T₁ and/or T₂ relaxivity.
 17. An apparatus forthe detection of an analyte in a liquid sample, the apparatuscomprising: a substrate; at least one nanoparticle at least partiallyimmobilized on the substrate, wherein the at least one nanoparticlecomprises a polymer matrix layer about a magnetic core, wherein thepolymer matrix layer has a specific volume that varies as a function ofa presence and/or concentration of the analyte in the liquid sample, andwherein a change in the specific volume of the polymer matrix layercorresponds to a change in an NMR-measured property of protons in theliquid sample in the vicinity of the at least one nanoparticle.
 18. Theapparatus of claim 17, wherein the substrate comprises a membrane;wherein the apparatus is implantable; and/or further comprising acontrolled release unit in communication with a sensing unit, whereinthe sensing unit comprises the substrate and the at least onenanoparticle, and wherein the controlled release unit dispenses asubstance into a body in accordance with instructions, the instructionsat least partially determined according to one or more signals from saidsensing unit, said one or more signals indicative of a presence,absence, or concentration of an analyte in the body.
 19. A smart devicefor delivery or release of a moiety into a body, the smart devicecomprising: a sensing element comprising at least one nanoparticle, saidnanoparticle comprising a polymer matrix layer about a magnetic core,said polymer matrix layer responsive to a condition of a volume of fluid(e.g., fluid of a body) in contact with said nanoparticle; an RF antennaconfigured to detect an echo RF signal from said volume of fluidfollowing RF excitation of said volume; a processor configured todetermine a measurement of said condition based at least in part on saiddetected echo RF signal; a stimulus emitter configured to emit astimulus in response to said measurement of said condition; and acontrol release element configured to release or deliver a moiety out ofsaid polymer matrix layer (e.g., and into said body) upon receiving saidstimulus.
 20. The smart device of claim 19, wherein at least part ofsaid smart device (e.g., said sensing element, said RF antenna, saidprocessor, said stimulus emitter, and/or said control release element)is implantable within said body; wherein said volume of fluid is in vivoor wherein said volume of fluid is ex vivo; wherein said moietydelivered out of said polymer matrix layer comprises one or more of thefollowing: an atom, an ion, a molecule, a compound, a catalyst, anenzyme, an electroactive mediator, an electron-pair donor, anelectron-pair acceptor, a lanthanide, an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent, a biological molecule, ametabolite of a therapeutic agent, a peptide, a polypeptide, a protein,a carbohydrate, a polysaccharide and/or insulin; wherein said conditionis a concentration of an analyte in said body (e.g., glucose); and/orwherein said smart device is configured to operate in an open loop. 21.A smart device for delivery or release of a moiety into a body, thesmart device comprising: a sensing element implantable within a body,said sensing element comprising at least one nanoparticle, saidnanoparticle comprising a polymer matrix layer about a magnetic core,said polymer matrix layer responsive to a condition of a volume of fluidin contact with said nanoparticle; a miniaturized NMR detectorfunctioning in conjunction with said sensing element to detect an echoRF signal from said volume of fluid following RF excitation of saidvolume; a processor configured to determine a measurement of saidcondition based at least in part on said detected echo RF signal; astimulus emitter configured to emit a stimulus in response to saidmeasurement of said condition; a control release element configured torelease or deliver a moiety out of said polymer matrix layer (e.g., andinto a body) upon receiving said stimulus; and a power source forpowering operation of said smart device.
 22. The smart device of claim21, wherein said smart device is configured to operate in a closed loop.23. A method for detection of an analyte in a sample, the methodcomprising: (a) providing nanosensors, wherein the nanosensors comprisemagnetic nanoparticles linked to an analyte or analog thereof, and oneor more binding moieties linked thereto, the binding moieties responsiveto said analyte or analog thereof; (b) providing a fluid sample andplacing the sample and the nanosensors in a container under conditionsand for a sufficient period of time to allow analyte in the sample tobind to and compete off the binding moiety from the nanosensor; (c)placing the container in proximity to an NMR detector; (d) measuring oneor more relaxivity parameters of the sample in the container; and (e)determining one or more attributes relative to the sample; wherein thenanoparticle, binding moiety and analyte or analog thereof linked to thenanoparticle are size optimized to confer optimal relaxationmeasurements.
 24. A method for detection of an analyte in a sample, themethod comprising: (a) providing a fluid sample and one or more bindingmoieties, the binding moieties responsive to a target analyte or analogthereof and placing the sample and binding moieties under conditions andfor a sufficient period of time to allow analyte in the sample to bindto binding moiety; (b) providing nanosensors comprising magneticnanoparticles linked to analyte or an analog thereof; (c) placing thepre-incubated sample and binding moiety and the nanosensors in acontainer under conditions and for a sufficient period of time to allowanalyte linked to nanosensors to bind to and compete off the bindingmoiety from the analyte in the sample; (d) placing the container inproximity to an NMR detector; (e) measuring one or more relaxivityparameters of the sample in the container; and (f) determining one ormore attributes relative to the sample; wherein the nanoparticle,binding moiety and analyte or analog thereof linked to the nanoparticleare size optimized to confer optimal relaxation measurements.
 25. Themethod of claim 23 or claim 24, wherein the attribute is selected fromthe group consisting of presence of the analyte, amount of the analyteand concentration of the analyte.
 26. The method of claim 23 or claim24, wherein the analyte comprises at least one member selected from thegroup consisting of a protein, a peptide, a polypeptide, an amino acid,a nucleic acid, an oligonucleotide, a therapeutic agent, a metabolite ofa therapeutic agent, RNA, DNA, an antibody, an organism, a virus, abacteria, a carbohydrate, and a polysaccharide.
 27. The method of claim23 or claim 24, wherein the binding moiety comprises an antibody or aconjugated antibody.
 28. The method of claim 27, wherein the antibody isa monoclonal antibody.
 29. The method of claim 23 or claim 24, whereinthe fluid sample is water, saline, buffered saline, or a biologicalfluid.
 30. The method of claim 29, wherein the biological fluid isblood, a cell homogenate, a tissue homogenate, a cell extract, a tissueextract, a cell suspension, a tissue suspension, milk, urine, saliva,semen, or spinal fluid.